technical.org

CyTOF

Buffer prep

Maxpar Cell Staining Buffer (request through flow core)

About 17 mL needed per sample

10 µM cisplatin (2X concentration)

Prepare a working solution of 10 µM cisplatin (2X concentration) by diluting the Cell-ID Cisplatin in Maxpar PBS. For example, add 2 µL of 5 mM stock to 1 mL of Maxpar PBS.

Fix I Buffer

100 uL per sample

Prepared by mixing 1 part MaxPar Fix I with 4 parts Maxpar PBS.

Barcode Perm Buffer

2 mL per barcode sample

10x stock, dilute to 1x using PBS

Foxp3 Fixation/Permeabilization working solution

Mix 1 part of Foxp3 Fixation/Permeabilization Concentrate with 3 parts of Foxp3 Fixation/Permeabilization Diluent

1 mL needed per sample

1X Permeabilization Buffer

8 mL needed per sample. Prepare using Maxpar Water from the 10x stock

1.6% FA solution (request through flow core)

Prepare a fresh 1.6% formaldehyde (FA, 10x) solution from the 16% formaldehyde stock ampule. Use a 1 mL Norm-Ject latex-free syringe and compatible 0.1 µm syringe filter to filter the stock formaldehyde, and then dilute 1 part of the filtered stock formaldehyde with 9 parts Maxpar PBS.

Intercalation solution (request through flow core)

Staining protocol (fresh tissue digest)

Wash the cell pellet with 5 mL of Maxpar PBS.

Cisplatin (live/dead)

If staining culture cells:

Wash the cell pellet with 1 mL serum free DMEM. Spin down at 300g 5min

Resuspend the cells in 100 uL of serum free DMEM.

Add 100 uL of 2x Cisplatin, mix well and incubate at room temperature for 5 minutes.

If staining digested tissue

Wash the cell pellet with 2 mL of Maxpar PBS.

Resuspend the pellet in 100 uL of Maxpar PBS.

Add 100 uL of 2x Cisplatin, mix well and incubate at room temperature for 5 minutes.

Quench the staining by washing with 1mL Maxpar Cell Staining Buffer. Pellet at 300g 5min.

Repeat wash with 1mL Maxpar Cell Staining Buffer. Pellet at 300g 5min

Cell pellets are ready for fixation and barcoding

Barcoding

Resuspend cell samples completely in 100uL of 1X Fix I Buffer and

incubate for 10 min at room temperature.

After 10 min, add 900 ul Cell Staining Buffer and spin at 800G for 5 min.

Resuspend cells in 1mL 1x Barcode Perm Buffer, centrifuge at 800g for 5min, aspirate supernatant, mix pellet

Resuspend pellet in 800 uL of 1x Barcode Perm Buffer

Resuspend each barcode in 100 uL of 1x Barcode Perm Buffer and transfer into samples. Mix thoroughly for uniform barcode staining. Record the barcode ID for each sample

Incubate at RT for 30 min. Mix after 15 min.

Centrifuge samples at 800g for 5min. Wash the samples twice with 2 mL Cell Staining Buffer. Pellet at 800g 5min.

Resuspend the pellet in 50 uL of Maxpar Cell Staining Buffer. Pool samples for surface staining.

Stain Cells With Surface Antibodies

Add 1 uL of FC block (per sample) and incubate at RT for 10 min

Add 50 uL of the 2X surface antibody cocktail (per sample).

Mix and incubate for 30min at room temperature on rocker.

Wash twice with 2 mL Maxpar Cell Staining Buffer. Pellet at 300g 5min

Stain Cells With Nuclear Antibodies

Add 1 mL of Foxp3 Fixation/Permeabilization working solution. Pipet mix to fully disrupt the pellet

Incubate for 30-60 minutes at 2-8°C.

Wash with 2 mL of 1X Permeabilization Buffer, pellet at 800 x g for 5 min at room temperature. Discard the supernatant.

Repeat the wash one more time

Resuspend pellet in residual volume of 1X Permeabilization Buffer. This is typically 50 uL after decanting.

Without washing, add 50 uL of 2x intracellular antibody cocktail

Incubate for 1 hour at RT.

Wash twice with 2 mL 1X Permeabilization Buffer. Pellet at 800g for 5min

Resuspend cell thoroughly in residual volume by gently vortexing after final wash/aspiration.

Fix Cells (Fresh Fix)

Add 1 mL of the 1.6% FA solution to each tube and gently vortex to mix well.

Incubate cells at room temperature for 10 min. Pellet at 800g for 5min

Stain Cells With Cell-ID Intercalator-Ir

Add 1 mL of intercalation solution to each 1–3 million cell sample and gently vortex. Ensure that cells are well resuspended.

Incubate the samples at room temperature for 1 hr or leave at 2–8 degree until analysis (48hrs - 96 hrs)

PCS9 AAV injection

Target 1x10^11 virus genome/mouse via retro-orbital injection stock AAV8-D377Y-mPCSK9 Lot 171009#24 Vector Biolabs Conc 3.2x10^13 genomecopy/ml = 3.2x10^10 gc/ul Dilute in sterile MEM based on excel sheet, inject 200 ul per mouse

Mouse breeding

Here are a few recommendations to help combat a colonies potential breeding depression:

1. Set additional pairs of breeders in the fall (ideally September or October) – this will help offset the potential reduced production from existing breeders. Even setting them now (in November) can help. Ideally, throughout the year, you should be rotating breeders so that you always have pairs that are being set up, are at the highest level of production and others that are getting ready to retire so that you are not retiring or starting all of your breeders at the same time.
2. Make sure that females are not bred until they are between 6-8 weeks of age and males until 8 weeks of age. Plan to retire breeders after about 6 months of active breeding, but make sure that you have a new pair that is producing before you retire your old breeders.
3. Give your breeders brown paper shred (ask your care tech for some). This has been shown to help mice build a better, warmer nest for their pups. Huts are generally not recommended. The thought is that the female fends off the male from inside a hut thus delaying or stopping breeding.
4. Cages should be low on a rack where it’s darkest and there is less traffic.
5. Avoiding handling breeding cages as much as possible. Manipulating/handling the cage (even if only to check for the presence of a litter) adds additional stress to those animals and can affect breeding productivity.
6. If possible, don’t mix your breeders and experimental cages on the same rack. The disruption of moving the experimental cages can adversely affect the breeders. Ideally, breeding cages should only be moved when regular cage changing occurs every two weeks.
7. Place breeders on racks as far away as possible from doors, ATS’s, equipment, noise, etc. Subtle vibrations can cause stress with mice and effect breeding behavior. There is also additional traffic in those areas.
8. Remove male mice for the weekend and replace them on Monday. A brief separation can make the heart grow fonder.
9. Taking the feces from a different male and placing into the breeding cage can often entice the male to breed due to their competitive nature.
10. Swapping males between cages. If they aren’t performing in the current environment, maybe a new one will help.
11. Set triad breeders. Two females and one male will often produce better results. Just make sure to keep a close eye on them. The male will need to be removed if a female drops a litter.
12. Finally, contact the Breeder Core for assistance. We give free consultations, we can manage your breeding for you or help on a special request basis.

[email protected]

ChIP protocol

Reagents needed

Buffers

Buffer	Volume/sample (mL)	# samples	total volume (mL)
Glycine	1	4.5	4.5
1x PBS	25		112.5
lysis	0.5		2.25
Dilution	3.8		17.1

Protease inhibitors (store in -80)

100 mM PMSF: 174 mg in 10 mL Isopropanol, use as 100x

10mg/mL aprotinin: 10mg in 1mL H2O, use as 10000x

1mg/mL pepstatin A: 10mg in 10mL of DMSO, use as 1000x

Notes

The protocol is optimized for 1.8 million cells in 10cm dish

Cross-linking and sonication

Cross-linking for cultured cells.

Add 312.5 ul of 32% formaldehyde in EMEM to every 10ml of cell culture medium

incubate at RT for 10 minutes

Quench the reaction by adding 1mL of 1.375 M Glycine per 10 mL of medium at room temp for 5 minutes

Wash twice with 10mL 1x PBS with protease inhibitors

Scrape the cells of the plate in 5ml of 1xPBS, wash the plate with additional 5mL of PBS

Pellet by spinning at 800g for 10 minutes

Cross-linking for sorted cells

cross-linking was done in sorted back cell mix (32% PFS volume = cell volume/32)

After adding glycine, spin at 800 G for 10 min at 4 degree

Wash once with 1xPBS with inhibitors, pellet

Resuspend the pellet with 450ul of SDS lysis buffer with protease inihibitors Incubate on ice for 10 minutes

Sonicate at 35% amp for 1 minute with 5 sec pulses and 30 sec rests in between. This ensured around 500bp fragments.

Incubate on ice for an additional 15 minute.

Centrifuge samples for 10min at 13000 rpm at 4 degree.

Collect supernatant (400uL). Lysate can be saved at -80 at this step after snap freezing.

If continuing protocol add 3.6mL of ChIP dilution buffer with protease inhibitor.

Save 20ul of diluted sample as input control.

Chromatin Preclearing and Immunoprecipitation

Add 75ul of Protein A Agrarose/Salmon Sperm DNA to sample incubate for 30 min at 4 degree with agitation Centrifuge 1000 rpm, 1min at 4 degree, collect supernatant Aliquote each sample into 2x 2mL (Antibody and Isotype control) Add antibodies into the supernatant incubate at 4 degree overnight with agitation

Next morning, add 60ul of Protein A Agrarose/Salmon Sperm DNA, rotate at 4 degree for 1 hour Centrifuge at 1000 rpm for 1 min at 4 degree, remove supernatant without distrubing the pellet

Antibody	Company	Cat#	Conc.	Amount
Anti-KAT3B / p300	Abcam	ab14984	1mg/ml	2ug
Anti-H3K4Me2	Millipore	05-1338	1 mg/ml	2ug
Anti-H3K9Ac	Millipore	06-942	1 mg/ml	3ug
Anti-H4Ac	Millipore	06-866	unpurified serum	4uL
Anti-acetyl Lysine	Abcam	ab21623		2ug
Anti-Klf4	rndsystems	AF3158	0.2 mg/ml	5ug
Anti-Klf4	Santa Cruz	sc-166238	2 mg/ml	2ug

Washes and Elution

Wash with the following buffer for 5 minutes 1x Low Salt 1x High Salt 1x LiCl 2x TE Buffer Prepare fresh elution buffer (1% SDS, 0.1M NaHCO3) Elute the complex by incubating with 250ul of elution buffer at room temp for 15 min, spin down and repeat 1 more time, combine supernatant Add 20ul of 5M NaCl to the elution (for input control, add 85ul of elution to the tube, then add 4.64 ul of NaCl), incubate at 65 degree for 4 hrs, store samples at -20 after. Thaw samples next morning, add 10uL of 0.5M EDTA, 20uL of 1M TrisHcl PH6.5, 2uL of 10mg/mL Proteinase K to each sample. For input control, add 2.32uL EDTA, 4.64uL Tris-HCl, 0.5uL Proteinase K. Incubate at 45 degree for 1 hour After the last step, IP samples should have about 550 ul of volumn, Input control samples should have 116uL.

Recover DNA with phenol/Chloroform extraction

Add one volume (550 uL/116 uL) of phenol:chloroform:isoamyl alcohol to the sample. Vortex for 20 sec Centrifuge at RT 5min, 16000 g. Transfer upper aqueous phase (~500uL/100uL) to fresh tube Add 1/10 (50/10 uL) volume of 3M sodium acetate (pH 5.0), 1uL of glycogen to samples Add 2 (1000uL/200uL) volume of 100% Ethanol Mix and freeze overnight at -20 degree (or 2hours at -80c) Centrifuge at 16000 g for 30 min at 4 degree Aspirate supernatant taking care with pellet. Add 1 ml 70% ethanol and spin at 16000 g for 15‟. Aspirate ethanol and remove remaining ethanol by pipet. Remove supernatant and dry the pellet with lid open at RT for 10-15 min Resuspend DNA in 30uL of H2O

Alternative Proceed with PCR purification kit (PB buffer needed 2750 ul and 580 uL)

Currently having issue with the column that little DNA can be recovered calculator for volume needed.

Sample number	volume for input	volume for IP	Total volume
7	580	2750	24975.

RNA immunoprecipitation

Notes:

The protocol is optimized for detection of protein-RNA interaction in the nuclei.

All steps should be performed on ice.

Cells seeded on T175 culture dishes at 70 – 90% confluency has been tested.

Buffers (prepare with nuclease free water)

hypotonic buffer (5mL per sample)

Component	TargConc (mM)	Stock Conc (M)	Stock Vol (uL)
Tris-HCl (pH7.5)	10	1	110
NaCl	10	1	110
MgCl2	2.5	1	27.5
TargVol(mL)	11	H2O	10.7525

RIP buffer (1mL per sample)

Component	TargConc (mM)	Stock Conc (M)	Stock Vol (uL)
Tris-HCl (pH7.5)	50	1	125.
NaCl	150	1	375.
MgCl2	1	1	2.5
IGEPAL CA-630	0.5%	10%	125.
EDTA	5	0.5	25.
DTT	1	1	2.5
RNaseOUT (u/mL)	200	40000	12.5
TargVol(mL)	2.5	H2O	1.8325

NT2 buffer Day1 0.5mL/sample

Component	TargConc (mM)	Stock Conc (M)	Stock Vol (uL)
Tris-HCl (pH7.5)	50	1	75.
NaCl	150	1	225.
MgCl2	1	1	1.5
IGEPAL CA-630	0.05%	10%	7.5
EDTA	5	0.5	15.
DTT	1	1	1.5
RNaseOUT (u/mL)	200	40000	7.5
TargVol(mL)	1.5	H2O (mL)	1.167

NT2 buffer Day2 5.5 mL/sample (x2 for target and IgG)

Component	TargConc (mM)	Stock Conc (M)	Stock Vol (uL)
Tris-HCl (pH7.5)	50	1	600
NaCl	150	1	1800
MgCl2	1	1	12
IGEPAL CA-630	0.05%	10%	60.
EDTA	5	0.5	120.
DTT	1	1	12
RNaseOUT (u/mL)	50	40000	15
TargVol(mL)	12	H2O (mL)	9.381

Step by step guide

trypsinze the cultured cells after treatment.

Resuspend the pellet in 5mL of hypotonic buffer with protease inhibitor (1:500). Incubate on ice for 15 min with periodic mixing.

Pellet nuclei by centrifugation at 2500g for 15 min at 4 degree.

Resuspend nuclei pellet in 1mL of RIP buffer with protease inhibitor (1:100),and passed through 25 5/8 G syringe 5 times (use 5mL syringe). Centrifugation (counter top) at 13000 rpm for 15 min at 4ºC.Collect supernatant.

To each sample, add 30 µl DNaseI (1 units/µl). Incubate the samples at 37°C for 10 minutes, with periodic mixing (via gentle tapping of the tube).

Mix the beeds well, transfer 50 uL into a Ep tube, pellet at 12000g for 20 sec, wash twice with 200 uL of NT2 buffer, remove supernatant.

After the 10 min incubation, place the samples on ice and collect 5uL of each sample as input (for western and qPCR).Add 250uL of Trizol to the samples for RNA and freeze at -80 degree.

Add the protein lysates to the washed beads, incubate for 30 min at 4 degree with rotation, centrifuge at 12000 g for 20 sec and collect supernatant (cleared lysate).

Divide the 1mL sample into 500 uL aliquots for antibody and IgG respectively.

Add antibody to the cleared lysate and incubate overnight at 4 degree.

Antibody	Company	Cat#	Conc.	Amount
Anti-Klf4	Santa Cruz	sc-393462 X	2mg/ml	10ug
Normal Mouse IgG	emdmillipore	12-371	1mg/ml	10ug

Add the lysate-antibody mix to 75 uL of washed beed and incubate at 4 degree for 2 hours.

Pellet the mix at 12000g for 20 sec, discard supernatnat, wash with 1mL of NT2 buffer for a total of 5 times.

During the final wash, save 10 uL of the beeds for western blot analysis.

After final spin, remove supernatant and add 1mL of TRIzol to the mix, resuspend and mix , incubate at RT for 5 min

Thaw the input sample and process for RNA extraction accordingly.

Add 200 ul of choloroform to the mix, vortex mix for 15 sec, incubate at RT for 3 min.

Centrifuge at 12000g for 15min at 4 degree.

Collect top aqueous phase into a new tube, add 0.5 mL isopropanol and 1.5ul of Glyco Blue. Mix and incubate at RT for 10 min.

Centrifuge at 12000g for 10 min at 4 degree.

Discard supernatant, wash the pellet with 1mL of 75% ethanol. Mix and centrifuge at 7500 g for 5 min at 4 degree.

Discard supernatant, dry the RNA with lid open for 10 min.

Resuspend the RNA in nuclease free water, incubate at 60 degree for 10 min with occasional pipet mix

Biotin-labeled RNA pull-down

Reference: Long Noncoding RNA as Modular Scaffold of Histone Modification Complexes

Nuclei lysate preparation.

Nuclear isolation buffer

1.28 M sucrose; 40 mM Tris-HCl pH 7.5; 20 mM MgCl2; 4% Triton X-100

RIP buffer

150 mM KCl, 25 mM Tris pH 7.4, 0.5 mM DTT, 0.5% NP40, 1 mM PMSF and protease Inhibitor

10^7 cells are resuspended in 2mL PBS, 2 mL Nuclear isolation buffer with 6 mL of H2O, keep on ice for 20 min with frequent mixing.

Pellet nuclei by centrifugation at 2500 G for 15 min (4 degree).

Resuspend nuclear pellet in 1mL RIP buffer, homogenize wiht 15-30 strokes. Centrifugation at 13000 RPM for 10min to pellet debris and collect supernatant.

in vitro Biotin-RNA transcription

Used AmpliScribe T7-Flash Transcription Kit (the biotin version is discontinued, need to find alternative).

Follow the company protocol with the following change

decrease the ATP, CTP and GTP to 1.5 uL (20 uL reaction) and add 1 uL Biotin-UTP (50mM) and 1 uL UTP

ApaI was used to linearize pCDNA3.1 containining Meg3 sequence

Purify the product using the Qiagen mini kit to remove DNA.

Use the RNA cleanup protocol and divide the reaction between two columns

Three micrograms of biotinylated RNA was heated to 90°C for 2 minutes, put on ice for 2 minutes, supplied with RNA structure buffer (10 mM Tris pH 7, 0.1 M KCl, 10 mM MgCl2), and then shifted to room temperature (RT) for 20 minutes to allow proper secondary structure formation.

Folded RNA was then mixed with 1mg of nuclear extract in RIP buffer and incubated at RT for one hour.

5% of the used lysate should be saved as input control.

Dynabeads preparation

Cat # invitrogen 11205D

Wash beeds (70uL for each sample), immobilize with using the DynaMag-2 Magnet

Wash with 100 uL Solution A (0.1M NaOH, 0.05M NaCl) twice for 2min.

Wash with 100 uL Solution B (0.1 M NaCl) once

Resuspend the beeds in equal volumes of Solution B and B&W buffer (2x 10mM TrisHcl pH 7.5, 1mM EDTA, 2M NaCl)

The volume of B&W should be 1/2 of the lysate volume.

Mix the RNA-lysate mix with the washed beeds, incuate for 30min at RT.

Separate the beeds with magnet for 2-3min

Wash 3 times with 1x B&W buffer

To dissociate the IP from the beeds, add 19.5 uL of B&W, 3uL of 1M DTT, and 7.5 uL 4x sample loading buffer, boil for 5min.

Separate the beeds with magnet and collect supernatant for WB analysis.

Western blotting

Gel casting with Bio-rad

Recipes for the gels

	Stacking	Resolving 7.5%	Resolving 10%	Resolving 12%
30% Acrylamide/bis (mL)	1.3	5	6.6	8
0.5M Tris-HCl, pH 6.8 (mL)	2.5	0	0	0
1.5M Tris-HCl, pH 8.8 (mL)	0	5	5	5
10% SDS (uL)	100	200	200	200
H2O (mL)	6.1	9.8	8.2	6.8
TEMED (uL)	10	20	20	20
10% APS (uL)	100	64	64	64

Recipes for 10x running buffer

Reagents	Conc	Weight
glycine	192 mM	72g
Tris	25 mM	15.15g
SDS		5g
H2O		500 mL

Recipes for 10x transfer buffer (Bjerrum Schafer-Nielsen)

Reagents	Conc	Weight
glycine	39 mM	29.3g
Tris	48 mM	58.2g
H2O		1000 mL

Ph should be ~9.2, Do not add acid or base to adjust PH.

For 1x buffer, add 100uL of 10x, 200 uL of Methanol to 700 mL of H2O

Recipes for TBS and wash buffers

10x TBS

Reagents	Weight
Tris-HCl	24g
Tris	5.6g
NaCl	88g
H2O	to 1L

PH should be around 7.6

Add Tween-20 to 0.1% in 1x TBS for the wash buffers

Blocking buffer

5% BSA in TBST

Antibody buffer

Dilute blocking buffer 1:10 in TBST

Antibody concentrations

Antibody	Company	Cat#	Host	Clone	MW	Dilution	notes
Goat Anti-Mouse HRP	jacksonimmuno	115-035-003	Goat			1:10000	Can try lower dilution
Goat Anti-rabbit HRP	Cell Signaling	#7074	Goat			1:2000
Donkey Anti-Goat HRP	Novex	A16005	Donkey			1:5000
Anti-β-Actin	Sigma	A5441	Mouse	AC-15	42 kDa	1:60000
Anti-GAPDH	millipore	MAB374	Mouse	6C5	38 kDa	1:4000
Phospho-Smad2/3	Cell Signaling	#8828	Rabbit	D27F4	52, 60 kDa	1:1000
Phospho-Erk1/2	Cell Signaling	#4377	Rabbit	197G2	42, 44 kDa	1:1000
Anti-Smad7	Santa Cruz	sc-365846	Mouse	B-8	46 kDa	1:600
Anti-KLF4	R&D Systems	AF3158	Goat		55 kda	1:2000	Some strong nonspecific bands
Anti-Klf4	Santa Cruz	sc-393462 x	Mouse	B-8	55 kda	1:6000	Some strong nonspecific bands
Anti-Klf4	Santa Cruz	sc-166238 x	Mouse	F-8	55 kda	1:6000
Anti-Klf4	Invitrogen	PA5-27440	Rabbit	Polyclonal	55 kda	1:1000	no diff between KO WT
Anti-aSMA	Abcam	ab5694	Rabbit		42 kDa	1:60000
Anti-HDAC1	Cell Signaling	#34589	Rabbit	D5C6U	62 kDa	1:1000
Anti-PKM2	Cell Signaling	#4053	Rabbit	D78A4	60 kDa	1:1000
Anti-HIF1a	Abcam	ab179483	Rabbit	EPR16897	110 kDa	1:1000
Anti-PTEN	Cell Signaling	#9559	Rabbit	138G6	54 kDa	1:1000
Anti-Ezh2	Cell Signaling	5246S	Rabbit	D2C9	98	1:1000
Anti-Cxcr7	Abcam	ab117836	Rabbit		41 kDa	1:1000	Not working for WB?
Anti-SMMHC	Abcam	ab125884	Rabbit		227 kDa	1:1000
Anti-Calponin 1/2/3	Santa Cruz	sc-28545	Rabbit		33-36 kDa	1:1000
Anti-Klf4	Cell Signaling	#4038S	Rabbit		65 kDa	1:1000
Anti-CXCR-7	Abcam	ab72100	Rabbit		42 kDa	1:200	4 - 6 µg/ml
Anti-Phospho-Akt (Ser473)	Cell Signaling	#4058	Rabbit		60 kDa	1:1000
Anti-Fibronectin	Abcam	ab2413	Rabbit			1:1000
Anti-PTEN	Cell Signaling	14642S	Mouse	D3Q6G	54 kDa	1:1000
Anti-Phosphoserine	Invitrogen	MA1-91608	Mouse	3C171	NA	1:500
Anti-Dnmt1	Abcam	ab188453	Rabbit	EPR18453	183 kDa	1:1000
Anti-AKT	Cell Signaling	9272	Rabbit		60 kDa	1:1000
Anti-Dnmt1	Cell Signaling	5032S	Rabbit	D63A6	200 kDa	1:1000
Anti-Tet2	Abcam	ab124297	Rabbit		223 kDa	1:250
Anti-Dnmt1	Invitrogen	MA5-16169	Mouse	60B1220.1	183 kDa	1:1000
Anti-Lamin A/C	Cell Signaling	#2032	Rabbit	Polyclonal	28, 70	1:1000

Drug Concentrations

EdU

Carbosynth, cat#: NE08701 Dissolve in saline on the day of use (don’t store) 5 mg/ml Calculate amount Mouse * 2 * 250ul Inject 50 mg/kg (1.25mg/mouse, 250ul) IP the day before harvest at 5pm Inject 50 mg /kg IP 8am (1hour before harvest) Harvest 9am

Additional reagents: Alexa Fluor™ 594 Azide: Catalog number: A10270 Click-iT® Cell Reaction Buffer Kit Catalog no. C10269

Tamoxifen

Recipe 0.5ml 100% EtOH 9.5ml Oil 100mg/10ml (1l) Use 100ul (1mg)/mouse/day for 5 days (Myh11) Use 125ul for 12 days for Gli1 mice

AngII

Target 1uM (relooked literature, 100nM might be more widely used) 25mg/ml (25g/L) MW 1046.19 25g/L = 0.023896 Mol/L =23.896mM To reach 100uM, 1:238.96 dilution 3+716.88 1:1000 for final dose

PDGF-BB

Millipore GF149

Note some lots were coming already reconstituted.

Reconstitute in 50ul sterile MQ to make 0.2mg/ml stock solution

Add 450ul 100mM Acetic Acid with 0.1% BSA to make 20ug/ml (1000x) working solution.

For the solvent:

57.5 uL of glacial acetic acid in 10mL of MQ, Add 10 mg BSA

2-DG

Note: EMEM (10-009-CV) has 1000 mg/L glucose (6mM). 2-DG ranging from 1-100 mM has been shown to inhibit SMC proliferation. Testing 20 mM concentration. 15 mM has been used in FLIM experiment.

2-DG was desolved in H2O at 2M concentration (100x). 2M glucose will be used as control

PTEN KD and control cells will be kept in 0;1% FBS media for 24 hours, then treated with glucose/2DG for 24 hours (qPCR) and 48 hours (WB).

5-azacytidine

in vitro

Target final concentration 10 uM

Stock concentration 10 mM (1000x) concentration in DMSO

Typical treatment scheme.

Plate in full serum 24 hrs.

Low serum for 24 hrs

5aza Pretreatment for 24 hrs

PDGF+5aza redosing for 24 hrs

in vivo

Target dose 2 mg/kg b.w. Inject 3 times (i.p.) per week.

Dissolve powder directly in sterile saline at 0.5 mg/mL

Prepare solution fresh for each injection

MG132

Stock solution 10mM in DMSO (Sigma #M7449)

Target concentration: 10 uM (1000x).

1 hour pretreatment before 5aza.

JQ1

In vitro dosing 1uM

Note, cells do not tolerate JQ1 well with 0.1% serum, therefore 1% was used. Normal time line

Seed cells (afternoon day1)

switch to 1% serum (morning day2)

JQ1 dosing (Noon day3)

JQ1 2nd dosing + Treatment (POGF) (Noon day4)

If doing ChIP, cells were harvested 24 hours later

ITF2357

Stock: 1mM in DMSO

Target conc 500 nM, Add 1:2000 directly to media in plate

TGFb (#7666-MB R&D Systems)

Stock conc. 50ug/mL in 4mM HCL with 0.1% BSA

4uL of 12N HCl, 12mg BSA in 12 mL of MQ, sterile filter.

Target conc. 5ng/mL, dilute 1:10 with media and use as 1000x

Simvastatin

With NaOH activation. Activation procedure (M. Sadeghi, et al., J. Immunol., 165, 2712-2718 (2000)

25 mg of simvastatin is dissolved in 500 μl of ethanol.

Then 750 μl of 0.1 N NaOH was added to the solution and subsequently incubated at 50 °C for 2 hours.

The pH was brought to 7.0 by 1M HCl (need about 80 uL), and the final concentration of the stock solution was adjusted to 10mM by adding 4.642 mL HBSS. The stock solution will be aliquoted and frozen at -80 degree.

Target concentration 0.5 uM (dilute stock 1:10 and use as 1000x solution).

Note prepare solvent control.

DMSO

dissolve at 100 mM concentration in DMSO (200,000x stock)

Dilute 1:200 in media with 0.1% FBS and use as 1000x stock (0.5 uM final concentration).

The 1000x stock might be cloudy.

Simvastatin in vivo delivery

Based on this paper

prepare methyl cellulose gel

****

DAPI

Invitrogen D1306 Stock solution (-20 degree): 1 mg/mL (2.86 mM) in DI/MQ water 100x Working solution (Fridge): 1 ug/mL in DI/MQ water

For IF staining

Dilute 2.86 mM stock to 300uM: 31.47uL DAPI + 268.53uL PBS

Dilute 300 uM intermediate solution 1:1000 in PBS to achieve working concentration.

100 mL volue is needed for the glass container for staining.

Stain for 5min in dark.

Wash 2-3 times with PBS before mounting.

Propidium iodide (PI)

Invitrogen P1304MP Stock solution (store at 4 degree, stable for 6 months): 1 mg/mL in DI/MQ water Use at 1ug/mL for flow cytometry (1:1000 dilution)

Recombinant Mouse Dkk-2

Reconstitute at 100 μg/mL in PBS containing 0.1% BSA

100 uL for 10ug

7 uL alliquots

Target conc. 25ng/mL, dilute 1:4 with media and use as 1000x

HMG-CoA Reductase Assay (Sigma CS1090)

Reagents preparation

1x Assay Buffer (190 uL per sample)

Dilute 5x stock with MQ water

NADPH reconstituation (4 uL per sample)

Reconstitute in 1.5 mL, aliquot and store in -80.

Sample preparation

Lysis buffer (for 60mm dish, try 100 uL per plate)

Component	TargConc (mM)	Stock Conc (M)	Stock Vol (uL)
Tris-HCl (pH7.5)	50	1	100
NaCl	150	1	300
MgCl2	1	1	2
IGEPAL CA-630	0.5%	10%	100.
EDTA	5	0.5	20.
PIC	1	100	20
TargVol(mL)	2	H2O	1.458

Measure lysate concentration and calculate the maximum amount input

***

Methylation-Specific PCR

CA injury

Use #6 strand for ligation. Do a single knot first, follow by one double knot When suturing, do double knots so it is not too tight.

Rimadyl

Stock: 50mg/ml, 50ug/ul Working conc. 5mg/kg Assuming 20g mice, need 100ug per mouse. Desired concentration: 100ug/100ul, 1ug/ul, diluted 50x

Cell culture

Rat Aortic Fibroblasts

Cell Biologics

Cat# RA-6075

1x10^6 P1

Preparation Conditioned Media from Cos7 cells

Expand Cos7 stably transfected with ShhN to 4x T175 flask.

Wash the cells 2x with HBSS and replace with 20 mL SM cell media + 0.1% FBS

Filter the conditioned media through 0.45μm syringe filter.

Aliquot the media and store at -80.

processing OCT tissue for IHC

thaw at rt for 15 minutes

DI water 5 minutes

PBC 5 minutes

100% methanol 10 minutes

0.3% H2O2 in methanol 10 minutes

0.05% tween-20/PBS 5 minute

PBS wash 5 minutes

block with 3% serum

antibody incubation overnight

Immunofluorescence on coverslip

Plate HepG2 in 24 well plate (put cover slip in wells) 0.18million/well. Let grow overnight After treatment (8hr), wash the cells with 500ul 1xPBS/well for two times. Fix the cells with 4% paraformaldehyde (15 min, room temperature) Wash with PBS 1x Methanol 10 min Permeabilize the cells with 0.5% tween (in PBS) for 5min at room temperature. Block the cells with 3% horse serum for 1 hour at room temperature. Dilute primary antibody in blocking buffer

Prepare petri dish with water-soaked filter papers (provide humidity) covered by a layer of parafilm

Pipet 50ul of diluted antibody on the parafilm and cover the coverslip onto the liquid drop. Incubate at 4 degree overnight.

Wash with TBST three times (10min room temperature shaking can use the initial 24 well plate)

Incubate secondary antibody (1:300 dilution with 1% BSA, 50ul/coverslip). Incubate 1hour at room temperature.

Wash with TBST 3 times (10minutes)

Mount and leave at 4 degree for 2 hours.

Take pictures.

Immunofluorescence for OCT sections

Thaw slides at RT for 15 min Rehydrate with dH2O, 5 min Post-Fix permeabilize 100% MeOH 10 min 0.05% Tween-20 in PBS 5 min PBS wash 5 min Block with 3% Horse serum in PBS 30 min Add anti GFP-FITC 1:200 in blocking buffer (300uL per slide)

antibody dilutions

Antibody	Company	Cat#	Host	Clone	Specificity	conjugation	Stock Conc(mg/ml)	Work Conc(ug/ml)	Dilution
Anti-Klf4	Santa Cruz	sc-166238 x	Mouse	F-8	HMR	none	2	2	1:1000
Anti-Klf4	Abcam	ab129473	Rabbit	Polyclonal	HM	none	0.8	1	1:800
Anti-Actin, α-Smooth Muscle	Sigma	C6198	mouse	1A4	HMR	Cy3			1:2000
Anti-GFP antibody	Abcam	AB290	Rabbit	Polyclonal	N/A	none	5		1:200
Anti-GFP antibody	Abcam	ab6662	Goat	Polyclonal	N/A	FITC			1:200
Anti-tdTomato	kerafast	EST203	Rat	EST203	N/A	none			1:200
Anti-CD34	Abcam	ab8158	Rat	MEC 14.7	Mouse	none			1:50
Anti-CD34	eBioscience	14-0341-82	Rat	RAM34	Mouse	none			1:200
Anti-Ly-6A/E	bd biosciences	553333	Rat	E13-161.7	Mouse	none			1:100
Anti-SCARA5	rndsystems	MAB4754	Rat	monoclonal	Mouse	none			1:20
Anti-Cxcr7	Abcam	ab117836	Rabbit	polyclonal	HMR	none			1:1000
Anti-PTEN	Millipore	04-409	Rabbit	A2b1	HMR	none			1:50
Anti-SMMHC	Abcam	Ab125884	Rabbit	polyclonal	HM	none	0.9	1.8	1:500
Anti-GLUT1	Abcam	ab115730	Rabbit	EPR3915	HMR	none	0.161	1	1:160
Anti-CD68	BioRad	MCA1957	Rat	FA-11	Mouse	none			1:50
Anti-HIF1a	Abcam	ab179483	Rabbit	EPR16897	HMR	none			1:500
Anti-PKM2	CST	#4053	Rabbit		HMR	none			1:100
Anti-Cxcr7	Abcam	ab72100	Rabbit		HM	none	1	10	1:100
Anti-Dnmt1	Abcam	ab188453	Rabbit	EPR18453	HMR	none			1:200

flow cytometry processing for cultured cells

Trypsinize to detach the cells Wash the cells twice with cold FA3 buffer Permeabilize with IC Fixation Buffer

Flow cytometry processing for tissue digest

Note

The protocol assumes staining in the format of a u-bottom 96 well plate.

After supernatant decant, about 50 uL volume is left in the wells

Buffer preparation

FA3 buffer

For surface staining and final FACS analysis,(sorting buffer) 1x PBS (Ca/Mg++ free), 1mM EDTA, 25mM HEPES pH 7.0, 1% FBS.

Intracellular Fixation & Permeabilization Buffer Set (cat. no. 88-8824)

IC Fixation Buffer is at 1x and ready to use. Prepare a 1X working solution of Permeabilization Buffer by mixing 1 part 10X concentrate with 9 parts distilled water.

Blocking

a. Have each samples (controls/samples) in 50 uL of FA3 buffer after single cell suspension preparation. b. Block the cells with anti CD16/CD32 (Fc block, Invitrogen, Cat# 14-0161-85, 100x) for 10 min at RT. Note Fc block should not be used with beeds. c. Proceed with staining without washing/spinning down.

Surface staining and viability dye

a. Prepare staining solutions by adding 1uL of antibody/Aqua viablity dye to 50 uL volume for each sample (e.g. if performing a 5 antibody staining for 3 samples, add 3 uL of each antibody + 3 uL of Aqua + 3 uL FC block in 129 uL of FA3 buffer) Note: I usually add 0.5 to the sample number to compensate any pipetting errors. b. Prepare staining solution for control (isotype/compensation) accordingly c. Add 50 uL staining solution to the 50 uL sample from blocking step d. Incubate at RT for 30 minutes (Protect from light). e. Spin down at 300 g for 5 minutes, wash twice with FA3 buffer f. Vortex the sample to fully dissociate the pellet after supernatant decant.

Fixing the cells

a. Fix the cells by adding 200 µL of IC Fixation Buffer to each well. b. Incubate at 4 degre for 1 hour, protect from light c. Centrifuge at 600 g for 5 min.Discard the supernatant. d. Wash with 200 uL of 1X Permeabilization Buffer e. Spin and discard supernatant

intracellular (cytoplasmic) staining

a. Prepare the staining solution similar as surface staining but with 1x Permeabilization Buffer b. Incubate the cell at 4 degree for 2 hours, protect from light c. Spin down and wash with FA3 buffer twice. d. Resuspend the samples in 300 uL of FA3 buffer for FACS analysis

Cell sorting with Sony MA900

System Startup

Turn on compressed air supply

Turn on hood

clean and wiping

Screw off the deflection plates, clean with kimwipe with 70% EtOH (never dry clean)

clean the interior of the sample collection, top to bottom, clean the door last.

Reinstall the deflection plates, hand tight, make sure it is stable.

Turn on the power using the POWER/STANDBY button on the front panel of the main unit. The LCD should say “standby”

Launch the sofware MA

Select maintenance

Select Ethanol Priming (NOT ethanol cleaning)

Follow onscreen instructions.

Ethanol priming

Check the liquid volumes.

Note: DI water bottle is filled with MQ in room 4208 Note: 70% ethanol: 490mL 200 proof ethanol fill up to 700 mL Note: Find cleaning chip from the drawer, load with label on top facing you.

Connect two tube from the slide wall together to bypass sheeth filter.

Connect blue line from sheath to A

Connect DI water line to B

Insert cleaning chip, leave packaging on the side in the chamber

Click next and start (takes ~5-8 min)

DI water rinse

Switch DI water line back from B to DI

start (takes 5-8 min)

Sheath rinse

reconnect sheath filter to original position

switch sheath line from A to sheath tank

start

account login page should show up once done.

login with lab account:

Username: WeiserEvansM Password: password1

Scan sorting chip with pop-up camera.

For our experiment, use 100 micron targeted mode (same for plate sort)

Store the cleaning chip back to the bag and into the drawer

Load the sorting chip with 100u mark on top facing you.

Select all lasers

Select standard configuration

Sheath Filter Debubble during the process. Check sample probe and make sure sheath fluid is dripping from the tip

Run Automatic Calibration

Note: always select sort calibration if sorting

Load 1 ml or more of undiluted, automatic setup beads in a sample tube, start a timer to record the calibration time.

Note: automatic setup beads is in cold room 4213. Vortex to mix, should be blue in color. Make sure it has at least 1mL volume. If not refill from the stock (one poping is the one in use).

Always select “Targeted”. 4k eps max

Fill in spreadsheet to record the start up readouts.

When finished,put the automatic setup beads back to cold room.

Turn on lights and temperature under cytometer tab.

Run probe wash and record the time to recover from blinking to solid green and record in excel.

Sorter should be ready now.

Sorting samples

Important notes:

Never close the software window with x unless done

Running analysis before sorting

Prepare about 10 mL of 10% bleach and 12 mL DI water for shutdown.

login with lab account:

Username: WeiserEvansM Password: password1

Select a previous experiment (left panel at the bottom).

Name the experiment

Click create new experiment (right, bottom).

Check fluid levels (bottom right or on the LED panel)

Double click the tube info to name the sample being sorted

Press the sample loader door button to open.

Remove cap and load sample tube with suitable adapter.

Press the button to close the sample loader door.

Sample stop condition (sorting or none)

recording stop condition (none)

Click start, Click record.

Click pause after accumulating some events

Adjust gate and compensation as necessary

Sorting

Note: never open the collection door during sorting

Place collection tubes into holder

Put the holder into the collection stage, close door

Click Load collection in the sorting control panel

select auto record, 5mL tubes, Normal.

Sort settings (gates and stop count)

Resume data acquisition

Sort Start, click stop once done.

Unload collection tubes.

Create a new tube and name the sample.

Run a bit of FA3/PBS between samples

start sorting the next sample

Print PDF by clicking the open area in the graphs, print PDF should show up on the ribbon.

run PBS at highest pressure. Probe wash

After all samples done, export database.

save database under C:\FCS\WeiserEvans

Shutting down

Shutdown –> Software and Hardware

Start

Bleach cleaning: Normal cleaning

use the 10 mL bleach prepared, should have 6 mL left after.

DI water rise: Normal cleaning, keep DI inside the sample probe

should have 6mL left after.

Shutdown ok

Wipe down the sample loader, collection area and collection stage with EtOH.

Refill sheath fluid, discard waster, shutoff compressor and hood.

Tissue digestion for AO and CA

Solutions Digestion solution: 3.2mg/ml collagenase II, 0.75mg/ml elastase (Worthington), 0.2mg/ml soybean trypsin inhibitor (sigma) in Hank’s buffered saline solution (HBSS), pH7.5. Serile filer with 0.2um syringe filter. FA buffer: 0.1% FCS in 1X PBS, sterile filer with .2um syringe filter. FA3 buffer (sorting buffer): 1x PBS (Ca/Mg++ free), 1mM EDTA, 25mM HEPES pH 7.0, 1% FBS or BSA (For 50ml solution: 5ml 10X PBS, 100ul 0.5M EDTA, 1.25ml 1M HEPES, 500ul FBS), sterile filer with 0.2um syringe filter.

Procedure: If doing matrigel, thaw aliquots on ice in fridge in the morning. Isolate aortas, carotid arteries from mouse. Wash the artery with HBSS once to remove serum from the medium. Tissues are minced with blade and transferred to digestion solution (tissue from 1 mouse need about 1ml) and incubate at 37oC in the incubator for 1 hr (do not exceed 1.5hr). Pipet mix every 10 min. Wash once with FA buffer (centrifugation: 1100 RPM, 12 min, 4oC, take care to remove all the fatty tissues at the surface of the supernatant). Pass the single cell suspensions through a 70um filter, pellet the cells again at the same setting. Antibody incubation: resuspend the cells in 100ul of FA buffer , add 3ul of Sca1APC antibody, and incubate on ice for 1 hour (minimum 45min). If doing matrigel injection, freeze some P1000 tips and Eppendorf tubes in -20. Spin down the cells and resuspend in 1ml of FA3 buffer. DAPI is used for staining cells (add right before sorting at flow core). Flow cytometry core is on the 4th floor, RC1 south.

Heparin 30IU/ml (stock 20kU/ml, sigmaH3393, 666.666X, for 700ul matrigel need 1.05ul) VEGF 100ng/ml (stock 50 µg/mL  in PBS R&D systems 493-MV/CF, 500x, for 700ul matrigel need 1.4ul) FGF2 500ng/ml (stock 100 μg/mL in PBS R&D systems 3139-FB-025/CF, 200x, for 700ul matrigel need 3.5ul) Note VEGF and FGF2 are ordered carrier free (BSA free) to prevent immune reaction from the host.

Mouse eye bleeding

heparinize eppendorf tubes and capillary tubes Dilute 20kU/mL Heparin 200x to 0.1u/uL Add 20uL of heparin into eppendorf tube. Run heparin through the capillary About 200uL whole blood will be needed for both TG and CHOL

Mouse fibroblast culture from ear snip

Digestion mix prep

6.25 mg/ml Collagenase, Type 4 (worthington LS004188) in HBSS

Need 0.5 mL per mouse

Tissue Harvest

Clean the ear area with iodine swab

Remove ear snip

Rinse the tissue in 70% ethanol

Rinse the tissue in 1x PenStrep in PBS

Tissue Digest

Mince the tissue into smaller pieces

Add 0.5 mL collagenase digestion buffer

Incubate at 37c for 25 minutes

Spin down for 5min at 1000 rpm with a countertop centrifuge

Discard supernatant

Wash once with 1 mL of HBSS and spin down

Add 0.5 mL 0.25% trypsin

Incubate at 37c for 20 minutes

Centrifuge and discard supernatant

Resuspend in 0.5 mL DMEM full medium

Triturate to break up cell aggregates and transfer into a 6-well plate

Add 2 mL of medium and place in the incubator.

Mouse Tissue Harvest

Heart

Perfuse from RV with 10 mL of 1.1% KCl to keep the cardiac tissue in dialated state for better morphology.

Mouse Tissue processing

Paraffin embedding

Ideally, Tissues should be in Formalin for 48 hours and 4 hours of 70% ETOH

Select program “Kidney Overnight”

Check reagents, level should be between Max and Min

Insert samples in the basket, best to use the middle rack.

Clean level sensor with a wipe

Hit Yes!

Next morning, check the tissue embedder is on and melted

Silent alarm

Press OK

Clean sensor

Standard cleaning

Tissue processing for spatial transcriptomics

cryoprotect with 10% DMSO in 1x PBS heparin

Perfuse the cardiac tissue with 1x PBS heparin with cold 10% DMSO

use regular perfusion protocol for heart. 3 mL through RV, 2 mL through LV, clamp AO, 5 mL through LV.

Keep at 4 °C for 10 min

Dry the excess liquid and proceed with fresh frozen protocol by 10x

recombinant Shh

3.5ug/mL for 48 hours to test Gli2/Gli1 protein expression use PDGF as control (should also induce Gli2)

Buffers and recipe

Proteinase K

solvent: 0.5mL 1M Tris-HCl(pH 8.0), 0.5mL 0.5M EDTA (pH 8.0), 4mL H2O Conc. 10 mg/mL

PBS-heparin

prepared 20ku/mL heparin stock solution. Add 1ml of the stock heparin to every 250mL of PBS and sterile filter.

37% Formaldehyde

prepare 1N NaOH Weigh 1.84g paraformaldehyde mix with 5 mL H2O and 175 uL of 1N NaOH Dissolve by incubating on the 60 degree heatblock. Cool the solution to RT Filter through a 0.45-μm syringe filter. Keep at RT for up to 1 week.

FA3 sorting buffer

Component	Target Conc (mM)	Stock Conc (mM)	Stock Vol(ml)
PBS	1x	10x	30
EDTA	1	500	0.6
HEPES(PH7.0)	25	1000	7.5
FBS	100%	1%	3
H2O	258.9	Total Vol	300

ImageJ

Overlay two images manually

Load the two images On first image, Image|Overlay|Add image… to add second image with X=Y=0, Opacity 33% Image|Overlay|To ROI manager…, select second image name in ROI manager window Select most any tool, e.g. segmented line tool, can then drag image2 over image1 to align Select None then causes the tool to do its drawing operation rather than dragging image2 Deselect image in ROI Manager window, then click on its name in ROI mgr, causes tool to again drag image2

Microscope

SHG

1. Deparafinize: 3X for 5 min in Xylene
2. Washes:

Rehydrate with ethanol washes for 3 min each: 100% ethanol 100% ethanol 90% ethanol 70% ethanol PBS PBS

1. Mount with no Dapi Vecta mount
2. Confocoal:

Cube: C051 in position1, mirror in position2 SL_SHG SL_SHG GFP for cotaininig with GFP BS SMP760 Laser Mai Tai on 800nm, 3% 700 gain

Cholesterol E kit (FUJIFILM/WAKO)

Dissolve color reagent in buffer solution (lasts 3 weeks at 4 degree) Create serial dilution for standard 200 mg/dL, 100 mg/dL, 50mg/dL, 25mg/dL in MilliQ Dilute Plasma 1:8 for assay Mix 2ul of sample/standard with 200uL of color reagent Incubate at 37 degree for 5 minutes Measure abs at 600 nm (and 700 nm)

Mouse CM isolation (Langendorff-Free)

Buffer preparations

Note 1: all buffers should be sterile filtered and good for 2 weeks at 4 degree protected from light

1. EDTA buffer (30 mL per mouse)

Component	TargConc (mM)	Stock Conc (M)	Stock Vol (mL)
NaCl	113	4.52	3.75
KCl	5	2	0.375
NaH2PO4	0.5	0.5	0.15
HEPES	10	1	1.5
EDTA	5	0.5	1.5
Component	TargConc (mM)	MW	Weight (mg)
Glucose	10	180.16	270.24
BDM	10	101.11	151.665
Taurine	10	125.15	187.725
TargVol(mL)	150	H2O	139.3
Ph to 7.8	approx	1N NaOH	0.75

1. Perfusion buffer (90 mL per mouse, accounting for Col and STOP)

Component	TargConc (mM)	Stock Conc (M)	Stock Vol (mL)
NaCl	113	4.52	3.75
KCl	5	2	0.375
NaH2PO4	0.5	0.5	0.15
HEPES	10	1	1.5
MgCl2	1	1	0.15
Component	TargConc (mM)	MW	Weight (mg)
Glucose	10	180.16	270.24
BDM	10	101.11	151.665
Taurine	10	125.15	187.725
TargVol(mL)	150	H2O	132.2
Ph to 7.8	approx	1N NaOH	0.75

1. Collagenase buffer (60 mL per mouse) in perfusion buffer Prepare right before procedure, warm to 37 degree

Component	TargConc (mg/mL)	Weight (mg)
Collagenase 2	0.5	125.
Collagenase 4	0.5	125.
Protease XIV	0.05	12.5
Volume (mL)	250

1. Stop buffer (10 mL per mouse)

Component	TargConc (%)	volumn
FBS	5	2
perfusion Buffer (mL)	40	38

Mouse Pre-treatments

One hour prior to tissue harvest, mice were IP injected heparin (200uL of 1,000 units/ml solution) (1,000 units/ml, 200ul per mouse)

Prepare the equipment (use 27 G needle)

1. 20 mL EDTA buffer, 10 mL perfusion buffer, 50 mL Collagenase Buffer in Syringes, keep the collagenase buffer warm with heatpad
2. Prepare 3 60mm dishes with 10 mL of each buffer

Tissue harvest

Induce and maintain anaesthesia with isoflurane Open chest and expose heart Move left lung and Cut inferior vena cava and decending AO Perfuse from right ventricle with 7 ml of EDTA buffer in 1 min Clamp Aorta with hemostat and cut to extract heart Proceed to tissue digestion in a 10cm dish with EDTA buffer

Tissue digest

1. Perfuse from the left ventricle with EDTA buffer (10mL in 6 min). Apply only enough pressure to the syringe to keep the heart fully inflated. Move heart to perfusion buffer
2. Using the same perforation, perfuse the heart with 3 ml Perfusion buffer to clear out EDTA. Transfer heart to plate with digestion Buffer
3. Perfuse with eyzyme digest cocktail (apply minimum pressure needed to keep heart full inflated, ~2 ml/min) Full digestion can be achieve with 25 mL to 60 mL depending on the condition of tissue/mouse. “Signs of complete digestion include a noticeable reduction in resistance to injection pressure, loss of shape and rigidity, holes and/or extensive pale and fluffy appearance at the heart surface, and ejection of myocytes into the effluent buffer, which are just visible to the naked eye.”
4. Remove clamp. Physically dissociate cardiac tissue in a new 10cm dish with 5 mL volume trituration with a 1000 µL pipette(cut tip).
5. Bring the volume up to 15 mL with stop buffer
6. Pass the cell suspension through a 250 µM mesh filter
7. Centrifuge at 50 g for 1min, remove supernatant and repeat 3 times
8. Collect supernatant, and pass through 70 um filter. Centrifuge supernatant at 400 g for 5 minutes to pellet non-myocytes non-myocytes will be subject to antibody labeling.
9. Combine the CM pellets and resuspend in 5 mL of Perfusion buffer three rounds of sequential gravity settling for 10 min. Combine pellet

Mouse CM isolation (based on Pinto Alexander 2016 Circ Res paper)

Buffer preparations

Note 1: all buffers should be sterile filtered and good for 2 weeks at 4 degree protected from light

1. EDTA buffer (20 mL per mouse)

Component	TargConc (mM)	Stock Conc (M)	Stock Vol (mL)
NaCl	113	4.52	5.
KCl	5	2	0.5
NaH2PO4	0.5	0.5	0.2
HEPES	10	1	2
EDTA	5	0.5	2.
Component	TargConc (mM)	MW	Weight (mg)
Glucose	10	180.16	360.32
BDM	10	101.11	202.22
Taurine	10	125.15	250.3
TargVol(mL)	200	H2O	194.65
Ph to 7.8	approx	1N NaOH	1

1. Perfusion buffer (20 mL per mouse, accounting for Col and STOP)

Component	TargConc (mM)	Stock Conc (M)	Stock Vol (mL)
NaCl	113	4.52	2.5
KCl	5	2	0.25
NaH2PO4	0.5	0.5	0.1
HEPES	10	1	1
MgCl2	1	1	0.1
Component	TargConc (mM)	MW	Weight (mg)
Glucose	10	180.16	180.16
BDM	10	101.11	101.11
Taurine	10	125.15	125.15
TargVol(mL)	100	H2O	91.1
Ph to 7.8	approx	1N NaOH	0.5

1. Collagenase buffer (10 mL per mouse) in perfusion buffer. Prepare right before procedure, warm to 37 degree

Component	TargConc (mg/mL)	Weight (mg)
Collagenase 2	3.2	32.
Soybean Trysin Inhibitor	0.2	2.
Elastase suspension (uL)	0.75	294.11765
Volume (mL)	10		q

1. Stop buffer (5 mL per mouse)

Component	TargConc (%)	volumn
FBS	5	1
perfusion Buffer (mL)	20	19

Mouse Pre-treatments

One hour prior to tissue harvest, mice were IP injected heparin (200uL of 1,000 units/ml solution)

Prepare equipment

20 mL EDATA buffer, 5 mL of perfusion buffer, 10 mL of digestion mix per mouse Prefill the syringes with 27 G needle

Tissue harvest (~ 20 min/mouse)

Euthanize with isoflurane Open chest and expose heart Move left lung and Cut inferior vena cava and decending AO. Perfuse from right ventricle with 7 ml of EDTA buffer in 1 min. Clamp Aorta and perfuse the left ventricle with 10 mL of EDTA buffer followed by 3 mL of perfusion buffer (try to use the same perforation).

Tissue digest (~ 1 hour 40 min)

Dissect the heart separate ventricle from atrium. Mince the tissue into 1-2 mm pieces and transfer to 10mL of digestion mix. Incubate at 37 degree for 50-60 minutes, trituration every 10 minutes. Filter the suspension through 250 uM filter to remove large debris Pellet the cells at 1100 RPM for 12 min, discard supernatant Resuspend the pellet in 10 mL of FA3 buffer, pass through 70 um filter Pellet the cells again and resuspend in 5 mL of FA3 buffer Pellet and remove supernatant. Resuspend the cells in 300 uL of FA buffer for antibody labeling

Staining and flow cytometry (~ 1 hour 20 min)

Add 3uL of anti-CD31-APC antibody to the cell suspension Mix well and incubate at 4 degree for 1 hour, protect from light. Add 1 mL of FA3 buffer, spin down and resuspend in 1 mL of FA3 buffer Add DAPI to the final concentration of 1 ug/mL (100x working solution) Sort back CD31 + population and CD31 - population Use 1x PBS (calcium and magnesium free) containing 2% FBS as collection buffer

Preparation for scRNA-seq submission

Buffer

1X PBS (calcium and magnesium free) containing 2% FBS. target concentration 900 cells/ul

Cell counting

Load both sides for the hemocytometer, count 4 sets of 16 corners on each side. Optimal concentration for cell counting is about 500-900/uL. Mix endothelial cells population with non-endo cells at the ratio of 1:9. Spin down and resuspend the cells at 900/uL (between 700 and 1200 / uL) in 1xPBS with 2% FBS.

Lung single cell suspension preparation

Tissue dissection

Perfuse left and right ventricle with 7 mL of PBS Hep.

Dissect lung tissue.

Mince the tissue with blade/scissor

Digest the tissue in regular digestion buffer with HBSS (1 lobe requires 10 mL of digestion buffer)

Filter with 250 uM filter

pellet and resuspend in 10 mL of FA3, Filter through 70 uM filter

Mix and spin down, wash with FA3 buffer one more time.

Pellet and proceed with staining

Transfection

293

24 well plate

well	6.5
Plasmid	DMEM	turbofect
1.07	100	2
6.955	650.	13.

protein related analysis done 48 hours post, RNA related can be done 24 post

Cos-7

10 cm dish

plate	1
construct	concentration (ng/uL)	Plasmid volume	DMEM (uL)	Turbofect
pCDNA3.1-ShhN	1547.95	3.8760942	600	12
linearized	939.6	6.3856960	600	12

for linearizing, 6.5 uL of plasmid (10ug) + 5 uL of NEBuffer3.1 + 10 uL of BglII (NEB R0144S) + 28.5 H2O. Digest at 37c for 2 hours Column purify after.

Lentiviral packaging and infection

Note, the protocol is for packaging pCDH-puro with pCMV delta R8.2 and pCMV-VSV-G

Day 1

Plate 7 x 10^5 293T cells in 4mL DMEM/10%FBS on a 60mm dish. Prepare one 60mm dish for each dish of target cells to be infected.

5.7 x 10^6 for T175 flask (40 mL)

293T stocks are in liquid nitrogen tank #2 Rack3 C

Day 2

Begin transfection ~24h later. For each transfection, place 200 uL sterile 150 mM NaCl into a 1.5 mL Eppendorf tube.

pCMV delta R8.2 1 μg

pCMV-VSV-G 0.2 μg

Target plasmid 1 μg

Add 8 uL TurboFect (Fermentas R0531).

Gently mix and incubate at RT for 15 min.

Add to 293T cells by gently dripping into the media. Incubate at 37°C overnight.

For T175 scale accordingly

NaCl 1629 uL

pCMV delta R8.2 8.14 μg

pCMV-VSV-G 1.63 μg

Target plasmid 8.14 μg

TurboFect 65.14 ul

Day 3

In the morning, aspirate media from the 293T cells and replace with 4 mL appropriate target cell media (e.g., RPMI/10% for A549 cells). Be careful not to disturb the 293T cells as they are loosely adherent.

Scale up for T175: 33 mL per flask

Plate target cells for infection the following day

AdvSca1-SM cells 1*10^6 cells/10cm dish

Day 4

Prior to infection, pretreat target cells with polybrene (Sigma H9268). 500X stock (4 mg/mL) prepared in sterile water is stored at 4°C in deli case. Add 2 uL of 500X stock per mL of target cell media (final concentration 8 ug/mL). Return to incubator for 30-60 minutes.

Pipet viral media from the 293T cells into 15 mL conical tubes.

Add 2 uL 500X polybrene stock per mL viral media. Filter through 0.45 um syringe filter.

Aspirate media from target cells and replace with 4 mL viral media.

Incubate at 37°C overnight.

Day 5

Repeat day 4

Day 6

Aspirate target cell media and replace with fresh media containing 2 ug/mL puromycin to begin positive selection of infected cells. (May need to perform kill curve with puromycin for different cell lines).

Days 7-13

Observe and expand resistant colonies as necessary

Validate overexpression

Create stock

Lentiviral media titration

Note: the protocol is modified based on Lenti-X™ qRT-PCR Titration Kit from takarabio.

Viral RNA extraction with trizol

Before starting, set heat block at 60 degree

Mix 100ul of viral media with 1mL of trizol reagent, incubate 5’ at room temperature.

Add 200 ul chloroform for every ml of TRIzol. Shake the samples vigorously for 15” to mix. Incubate at room temp. for 2-3’. Centrifuge samples for 15min at 12,000xg at 4 degree. (Centrifuging cold yields better phase separation).

RNA is in the aqueous phase. Aqueous phase is the colorless upper phase which corresponds to about 60% of the volume of TRIzol used. (DNA and proteins are in the interphase and the organic phase).

Transfer the aqueous phase to a fresh tube and precipitate the RNA by mixing with 0.5 ml of isopropanol for every ml of TRIzol used initially. Add 1ul of glycogen. Incubate at room temperature for 10’. Mix the tube by inverting and centrifuge for 10’ at 12, 000xg, at 4 degree.

Remove the supernatant. Wash the RNA pellet with 1ml. 75% Ethanol for every ml TRIzol used. Mix by flicking and inverting the tube and centrifuge at 7500xg for 5’ at 4 degree.

Remove the supernatant and dry the RNA by vacuum or air for 5-10’. Do not centrifuge when during under vacuum and do not overdry the RNA pellet. (When sufficiently dried, the pelet should appear whitish. If pelet is clear, it indicates that it has been overdried).

Resuspend the RNA in 50 uL RNAase free MQ water. To completely redissolve the RNA pelet, incubate the tube at 60C for 10’ with occasional pipetting up and down in between.

DNase I treatment (Thermo, #EN0521)

RNA	12.5 uL
10x reaction buffer	2.5 uL
50 mM EDTA	2.5 uL
DNaseI	2.5 uL
H2O	5 uL

incubate at 37 degree for 30min and 65 degree for 10 min

Revere transcription

Try measure the RNA with nanodrop. The current batch has around 20-40 ng/uL concentration.

For the RT reaction, use 12 uL from the DNaseI treated samples, 4uL of RT mix and 4 uL of H2O

2ul of RT reaction was diluted 10x (20uL) as 1x in qPCR (3uL was used in each reaction). 10x serial dilutions were performed. For the current batch, a standard (DNA) ranging from 5*10^4 copies/uL to 5 copies/uL fits the samples well.

In vivo lentivirus delivery

Protocol is based method described by Liu et al 2021 and Strappe et al 2005

Pluronic F127 (Sigma, # P2443) was dissolved in PBS overnight at 4 °C on a roller to achieve concentrations of 20% (W/V). Once dissolved the solutions were stored at 4 °C.

**

Creation of Stable Cell Lines

Determining antibiotic sensitivity

Plate cells at 0.1 million/well in 6-well plate.

Allow cells to adhere overnight.

Next morning, change medium to antibiotic containing medium.

Replenish selective media every 3-4 days

Observe cell survival over time (can be as long as 2-3 weeks)

Ex vivo culture of AdvSca1-SM cells

Culture media

alpha MEM (with GlutaMAX)

10% MSC qualified FBS

Freshly sorted cells were maintained in 20% FBS until ready to pass.

After P1 the FBS is reduced to 10%

1x Penicillin Streptomycin

Freshly sorted cells were maintained in 2x PS for the first 3 days.

1ng/mL murine basic fibroblast growth factor

R&D SYSTEMS # 3139-FB. Resuspend in sterile PBS w 0.1% BSA at 100ug/mL, 5uL needed per 500 mL media.

5ng/mL murine epidermal growth factor

R&D SYSTEMS # 2028-EG. Resuspend in sterile PBS at 200ug/mL, 12.5 uL needed per 500 mL media.

rShhN (try 4ug/mL, 400ng/mL, 40ng/mL, 4ng/mL, 0ng/mL)

R&D SYSTEMS # 461-SH. Resuspend in sterile PBS w 0.1% BSA at 100ug/mL. (20 ul aliquots made). Note, no rShhN is used at the moment.

Gelatin coating

Dilute the 2% Gelatin to 0.1% with TC grade water (2.5 mL + 47.5 mL H2O)

Add 1.0 mL of 0.1% Gelatin Solution per 10 cm2 of culture surface area (e.g., 350 uL per well if using a 12-well plate).

plate	Volume of Gelatin per well(uL)
24-well	300
12-well	350
6-well	960
60mm	2150
100mm	5670
T175	17500

Rock culture flask to coat surface; place in a 37°C incubator (with or without 5% CO2) for at least 30 minutes, and up to overnight.

Aspirate the excess gelatin solution from the culture flasks using sterile technique. Wash the plate with HBSS.

For freshly sorted cells, add 5.0 mL of complete growth medium per 25 cm2 of culture surface area (e.g., 700 uL per well if using a 12-well plate, 1.5 mL per well in 6-well plate). Place the gelatin-coated flasks in a 37°C, 5% CO2 incubator for at least one hour to equilibrate before inoculating with the cell suspension.

Cells were seeded at the density of ~20,000/cm2.

Primary culture (P0) will be maintained in AdvSca1-SM media (MEM α (with GlutaMAX) with 20% MSC qualified FBS, 2x Penicillin Streptomycin, 1ng/mL murine basic fibroblast growth factor, 5ng/mL murine epidermal growth factor) to reach confluency.

The cells will be expanded and maintained with full AdvSca1-SM media with reduced FBS (10%) and Penicillin Streptomycin (1x).

Generally, a confluent 6-well well can be passed into 1x 100mm dishes (P1), and then to 6x 100mm dishes (P2).

One confluent 100cm dishe can be divided into two cryovials for long term storage.

Ref

Cell stocks in liquid nitrogen tanks

SMC WT Tank1 6G

SMC PTEN KD Tank2 4B

AdvSca1-SM Tank2 4D

293T Tank2 3C

Kidney and Cardiac tissue for spatial transcriptomics

Note: this is just a overview of the 10x protocol, follow the original protocol for more details.

Fixation, staining and imaging

Prepare the following items before start

40 mL Methanol in 50 mL tube, chill to -20c (1 hour?)

Place a thermocycler adaptor on a thermal cyccler set at 37oc (5min)

Prepare 2x blocking as listed in the protocol

Prepare 3x ssc

If not coverslipping, only 50 ml of 3x SSC will be needed

3x SSC	Stock	final	50mL	550ml
ssc stock	20x	3x	7.5	82.5
Water	-	-	42.5	467.5

Mounting medium

Have a box of dry ice ready for transfer

Procedures

Remove slide from -80 and place on dry ice in a sealed container

Place the slide on the thermocyler adaptor with active surface facing up. Incubate 1 min

Wipe excess lipid on the back of the slide

Move slide to chilled methanol, close cap. Incubate 30 min at -20

During the incubation prepare the following (see protocol for details)

1x blocking buffer (vortex)

wash buffer

Antibody solution (if doing one step staining, goto 1.4, if require 2ndary ab, use 1.3)

Note Recommended DAPI is at 1 mg/mL, we have the same in -20

Place the slide in the slide cassette, active surface facing well

Add 70ul of 1x blocking buffer to each well, incubate 5min RT

Remove blocking buffer, add 50uL of antibody solution to each well, incubate 30min RT

Remove antibody solution.

Wash each well with 100 ul wash buffer, total of five washes

If mounting

Remove the slide from cassette

Dunk the slide 20x in 3x SSC in 50 mL tube

Wipe excess liquid from the back of the slide, don’t touch active surface

Add 200 uL mounting medium each slide. Apply cover slip. Drain excess

Proceed to imaging

After imaging, unmount the coverslip with 3x ssc

Place the slide in clean slide cassette

Add 50 uL of wash buffer to the wells

Proceed to Gene Expression

If not mounting (for permeabilization optimization)

leave the last wash in the well, proceed to Tissue optimization

permeabilization

Prepare the following

Resuspend permeabilization enzyme in 1.2 mL HCl.

Put the Permeabilization Enzyme at 37 degree (equilibrate for 15 min, no longer than 20min).