TITLE: Processing Human Colon muscle layers for Single Nuclei RNA-seq using PHOX2B antibody immunoselection
AUTHORS: Christina M Wright,  Robert O Heuckeroth

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">ABSTRACT</div><div class = "text-block">INTRODUCTION: The human enteric nervous system (ENS) is a complex network of neurons and glia that extends throughout the length of the bowel. There are many neuron and glia types, but very little is known about the human ENS compared to other species. There are two main ENS layers. The myenteric plexus neurons are clustered into ganglia with thick nerve fiber bundles running between ganglia. Submucosal neuron ganglia are scattered throughout the region between circular muscle and the epithelial lining of the bowel. One key problem with studying human ENS is that these cells are a very minor component of the bowel wall (< 1: 10,000 cells) and they are surrounded by other tissue. The bowel wall is not easily dissociated making it challenging to isolate cells of the human ENS for any type of analsysis. 


OBJECTIVES: To enrich for neurons from human colon myenteric plexus using PHOX2B antibody and FACS 


METHODS: 1) Human colon muscle was dissected from the bowel wall and then frozen in OCT. 2) Frozen muscle was sectioned on a cryostat to generate small fragments and disrupt muscle and connective tissue. 3) RNA quality was assessed from a few sections using an Agilent Bioanalyzer. 4) Nuclei were obtained from frozen sections using Dounce homogenization and stained with antibody to PHOX2B. 5). Nuclei were separated from other cell debris by FACS. 6) RNA-seq data were obtained using the 10X Genomic single cell sequencing platform.


RESULTS: This procedure worked well to isolate nuclei from human colon muscle. PHOX2B antibody labeling led to a small population of nuclei that appeared to be antibody stained under the microscope and have attached antibody by FACS. Unfortunately, single nucleus RNA-seq demonstrated that we had not enriched for myenteric neurons with this approach. RNA-seq data were obtained from many other cell types within the muscle layers of the bowel wall.


CONCLUSION:  This approach generated valuable data at a single nucleus RNA-seq level for many cell types within the muscle wall known to control bowel motility, but did not enrich for neuronal nuclei, even though PHOX2B antibody selectively stains myenteric plexus nuclei within the human bowel myenteric plexus

</div></div>

[STEPS]
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Half hour before the tissue arrives:Spray off forceps and scissors with RNase Away, and thoroughly rinse/soak in waterFetch dry ice.In the fume hood, add methylbutane to a petri dish on the dry ice. Place a few pieces of dry ice into the methylbutane to chill it.
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Transfer tissue, on ice, from the hospital’s Pathology unit to your laboratory in sterile ice cold 1x PBS in a 50 mL conical or similar container. (~30 minutes)
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Upon arrival to lab, don appropriate personal protective equipment (PPE) = (gloves, lab coat, face mask, face shield).
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Pour cold 1x Dulbeccos Phosphate-Buffered Saline and the tissue into a new sterile dish.
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Quickly trim off surrounding fat using Dumont #2 forceps and a fine scissors.
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Then, using the fine scissors and Dumont #2 forceps, carefully cut submucosa and epithelium off tissue, leaving only muscularis.
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Cut the muscularis into small pieces (~5 x 5 mm).
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Label the appropriate number of cryomolds with sample number and “muscularis+plexus.”
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Transfer the smaller tissue muscularis tissue pieces into Cryomold Biopsy specimen molds half-filled with RNase Free Tissue-Plus O.C.T. Compound. Add O.C.T. to cover the tissue.
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Place the cryomold containing the cold O.C.T.+plexus in the 2-Methylbutane in the fume hood.
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Wait 5 minutes for it to freeze. Then transfer to a -80° C freezer in appropriate containment (e.g. plastic bags in labeled boxes).
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Record human subject information linked to colon specimen.
?. [Human colon processing to collect and freeze muscle layer in O.C.T]
Autoclave tools for future use.
?. [Sectioning frozen human colon muscle layers on cryostat]
Fill an ice bucket with dry ice.
?. [Sectioning frozen human colon muscle layers on cryostat]
RNaseZAP a Dumont #2 Forceps. Rinse with deionized MilliQ water.
?. [Sectioning frozen human colon muscle layers on cryostat]
Transfer tissue labeled “Plexus” in biopsy cryomold on dry ice to Cryostat set to -20 °C.
?. [Sectioning frozen human colon muscle layers on cryostat]
Freeze the tissue onto a metal cryostat mounting device using a minimum of O.C.T. compound.
?. [Sectioning frozen human colon muscle layers on cryostat]
While waiting for tissue to freeze, label 3 DNA LoBind Tubes: “  Muscularis+Plexus”, “ Muscularis+Plexus”, and “  Muscularis+Plexus RNA”. Place these on dry ice or in the 20 °C for at least 3 minutes before sectioning so they have time to cool.
?. [Sectioning frozen human colon muscle layers on cryostat]
Place Dumont #2 Forceps in cryostat to chill.
?. [Sectioning frozen human colon muscle layers on cryostat]
Mount the tissue, insert the blade, and begin making 100 micron sections.
?. [Sectioning frozen human colon muscle layers on cryostat]
Using the Dumont #2 Forceps, collect all sections in “ Muscularis+Plexus” tubes EXCEPT 2-5 sections. Put 2-5 random sections in tube labeled “ Muscularis+Plexus RNA”.
?. [Sectioning frozen human colon muscle layers on cryostat]
Return the tubes with tissue to the -80°C on dry ice.
?. [Extracting RNA for BioAnalyzer Analysis]
Remove tubes labeled “RNA” from the -80. There should be one each for plexus and for surrounding muscle.
?. [Extracting RNA for BioAnalyzer Analysis]
Add 350 uL of buffer RLT plus to each tube.
?. [Extracting RNA for BioAnalyzer Analysis]
Homogenize the lysate by vortexing for 30s.
?. [Extracting RNA for BioAnalyzer Analysis]
Run through gDNA spin column. Centrifuge for 30s at >=8000xg – I did 10,000g this time. Last time 8,000.
?. [Extracting RNA for BioAnalyzer Analysis]
Discard the column and keep the flow-through.
?. [Extracting RNA for BioAnalyzer Analysis]
Add one volume (usually 350 uL of 70% ethanol to the flow-through and mix well by pipetting up and down 4 times. Do not centrifuge, and proceed immediately to next step.
?. [Extracting RNA for BioAnalyzer Analysis]
Transfer sample, including any precipitate that may have formed, to an RNeasy MinElute spin column placed in a 2 mL collection tube (find RNeasy columns in 4 degrees C freezer B). Close lid and centrifuge for 15s at > 8,000 g. Discard the flow thru
?. [Extracting RNA for BioAnalyzer Analysis]
Add 700 uL of buffer RW1 to the RNEasy MinElute spin column. Clsoe the lid and centrifuge for 15s at 8,000 g (10,000 RCF). Discard the flow-through.
?. [Extracting RNA for BioAnalyzer Analysis]
Add 500 uL Buffer RPE to the RNeasy MinElute spin column. Close lid gently, and centrifuge for 15s at >8000 g to wash the spin column membrane.
?. [Extracting RNA for BioAnalyzer Analysis]
Add 500 uL of 80% ethanol to the RNEasy MinElute column. Close the lid and centrifuge for 2 minutes at 8,000 g (10,000 RCF) to wash the spin column membrane. Discard the collection tube with the flow-through.
?. [Extracting RNA for BioAnalyzer Analysis]
Place the RNeasy MinElute spin column in a new 2 mL collection tube (supplied). Open the lid of the spin column, and centrifuge at full speed for 5 minutes to dry the membrane. Discard the collection tube with the flow-through.
?. [Extracting RNA for BioAnalyzer Analysis]
Place the RNeasy MinElute spin column in a new 1.5 mL collection tube (supplied). Add 14 uL of RNase free water directly to the center of the spin column membrane. Close the lid gently and centrifuge for 1 minute at full speed to elute the RNA.
?. [Extracting RNA for BioAnalyzer Analysis]
Analyze RNA on the Agilent RNA 2100 Bioanalyzer with the Agilent RNA 6000 Pico Kit, along with any additional samples.
?. [Extracting RNA for BioAnalyzer Analysis]
Follow the instructions on the Qiagen RNeasy Micro Kit for this section
?. [Preparing to isolate nuclei for RNA-Seq]
Prepare Hoechst, 2.5 mg/mL:-Dilute 10 mg/mL Hoechst 1:4 in nuclease-free water.-Using 3 mL syringe and 0.22 micron filter, filter the Hoechst.-Store at 4 degrees C.
?. [Preparing to isolate nuclei for RNA-Seq]
Prepare Normal Donkey Serum for blocking:- Normal Donkey Serum in 10 mL of sterile 1x PBS.-Filter the Normal Donkey Serum using a 3 mL syringe and a 0.22 micron filter.
?. [Preparing to isolate nuclei for RNA-Seq]
Prepare Diluted Nonidet P40 substitute:-Add 1 mL of RNase free Nonidet P40 substitute to 9 mL of RNase free water.-Place on rotator for half an hour to dissolve Nonidet P40 substitute.
?. [Preparing to isolate nuclei for RNA-Seq]
Autoclaving:-Rinse large iridectomy scissors and Dumont #2 forceps with RNaseZAP followed by MilliQ water. -Place in small autoclave bag.-Autoclave before use
?. [Preparing to isolate nuclei for RNA-Seq]
Reconstitute PHOX2B antibody:-Reconstitute antibody as per the manufacturer’s instructions. -500 µL of sterile, RNase free PBS was added to 100 µg of antibody, to make a final concentration of 0.2 mg/mL.-Aliquot the antibody into 50 µL aliquots and store at -20 °C until use.
?. [Preparing to isolate nuclei for RNA-Seq]
Schedule FACS sort:-Schedule FACS sort on MoFlo Astrios or an equivalent sorter with a 70 micron nozzle and the ability to detect DAPI/Hoechst.
?. [Preparing to isolate nuclei for RNA-Seq]
Schedule 10x Genomics Sequencing run:-Schedule 10x Genomics Sequencing run since samples will be analyzed on the day nuclei are isolated
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Get a large ice bucket.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Prepare the appropriate quantities of Lysis buffer and Staining buffer. Place them on ice.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Precool the Dounce homogenizer and pestles on ice.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Rinse large iridectomy scissors and Dumont #2 forceps with RNase Zap. Rinse with MilliQ water. Precool large iridectomy scissors and Dumont #2 forceps on ice in an open 50 mL conical.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Precool large and small centrifuges to 4 C.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Place a 6-well plate on ice. Fill wells with 6 mL of Lysis buffer.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Get dry ice. Transfer sectioned human tissue to the bench on dry ice.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Approximately 1 hour before FACS sort, put on appropriate PPE.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Use Dumont #2 forceps to transfer one Eppendorf tube with sectioned human tissue into 6 mL of Lysis buffer on 6 well plate.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Chop rapidly with large iridectomy scissors for 1 minute.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Using a 1000 uL pipet, transfer 2 mL of Lysis buffer + tissue/O.C.T. into a pre-cooled Dounce homogenizer.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Homogenize tissue with 20 strokes of the loose pestle and 50 strokes of the tight pestle.a.	Note: homogenization often induces suction and the formation of foam. Cover homogenizer with a paper towel and wear appropriate lab coat and PPE during this step to avoid getting splashed. Suction is particularly likely to occur for these tissue pieces because there are so many muscle fibers.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Put the tissue through a MACS SmartStrainer (30 micron) resting atop a 50 mL conical.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Chase with 2 mL of Lysis buffer to rinse the strainer. Switch out strainers if they appear to be getting clogged, between patients, or after 3 uses.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Repeat steps 9-14 until all Eppendorf tubes with tissue from the patient have been homogenized. Homogenized nuclei from the same patient may be combined into the same 50 mL conical.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
If nuclei from a second patient are being run that day, repeat steps 6-15.a.	Note: Since homogenization and FACS sorting takes a long time, we never ran nuclei from more than 2 patients in a single day to maximize RNA quality.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Spin down Lysis buffer+nuclei+OCT on large centrifuge at 590 G for 8 minutes at 4 degrees C.  Be sure to appropriately balance samples.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
After the centrifugation is over, use a Pipet-Aid to remove liquid from each 50 mL conical. Get the last bit of liquid out using a P1000.a.	Note: you probably will not see a pellet at this stage. Be very, very careful not to pipet out the nuclei at the bottom of the 50 mL conical. I typically tilt the 50 mL conical to the side and leave a small quantity of liquid at the bottom.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Resuspend nuclei from each patient in 990 mL staining buffer by pipetting 5-10 times with a P1000.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Add 100 µL of Normal Donkey Serum for blocking.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Add 1 µL of 2.5 mg/mL Hoescht.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Leave on ice for 15 minutes to block.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
For each patient, label 3 Eppendorf tubes:a.	Isotype control tubeb.	Phox2b tube #1c.	Phox2b tube #2
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Transfer 330 µL of nuclei in blocking solution into each tube.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Add 3.3 µL (3.3 µg) of goat IgG to the isotype control tube.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Add 16.5 µL of Phox2b antibody (3.3 µg) to Phox2b tubes #1 and #2.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Attach Eppendorf tubes to Glas Col Rotator with rubber bands. Rotate samples at speed 30 in the cold room on a rotator for 45 minutes.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Add 1 mL of 1x PBS (rinse buffer) to each tube.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Invert the tubes ~5 times to wash
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Spin in an Eppendorf centrifuge for 5 minutes at 400 RCF at 4° C.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
While spinning, prepare 1,300 µL of 1:400 Alexa Fluor 647 Donkey anti-goat secondary antibody in staining buffer.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
When spin is over, pipette out supernatant without disturbing the pellet.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Resuspend the pellet in 400 µL of secondary antibody in staining buffer.a.	Note: pipette sample 5-10 times until any residual clumps are gone.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Rotate samples at speed 30 in the cold room rotator for 30 minutes.a.	While waiting, label 1.5 mL Eppendorf tubes, 1 tube per patient. Add 5 uL staining buffer to the bottom of each tube. These are the collection tubes.b.	Also label Falcon round-bottom tubes, one tube per patient. These tubes will hold the sample to be sorted.c.	Place labeled 1.5 mL Eppendorf tubes and Falcon round-bottom tubes on ice to prepare for transport to FACS sorter.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
When 30 minutes is up, add 1 mL of 1x PBS (rinse buffer) to each tube with nuclei+secondary.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Invert the tubes ~5 times to wash.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Spin in an Eppendorf centrifuge for 5 minutes at 400 RCF at 4° C.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Resuspend samples in 1 mL of staining buffer.a.	Note: pipette sample 5-10 times until any residual clumps are gone. This is especially important to prevent clogging of the FlowMi strainer.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Pipette the liquid into a P1000 pipette tip and attach the FlowMi strainer to the base. Strain through the FlowMi strainer into a spare Eppendorf tube.a.	Note: if the FlowMi strainer clogs and liquid is not moving, do not panic! Slowly raise the plunger on your P1000 until it is fully raised. While holding the P1000 pipette tip, carefully remove the pipette tip from the P1000 pipettor. Then try to remove the FlowMi strainer from the base of the pipette tip with the lid of your Eppendorf while still holding the pipette tip securely. If you manage to do this successfully, pipette the liquid in the P1000 5 more times to thoroughly disrupt clumps, and try again with a new FlowMi strainer.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Pipette the liquid into a new P1000 pipette tip. Strain through the top of the Falcon round-bottom tube by placing pipette tip flush against the strainer lid.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Bring the samples and collection tubes to FACS on ice.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Run isotype control sample first to determine appropriate gating.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Sort Hoescht+, Phox2b+ nuclei (with higher signal than isotype control) into the collection Eppendorf tubes with 5 uL staining buffer using 70 micron nozzle (this can sort up to 1,000 nuclei per microliter).a.	Below is a sample image from a FACS sort. R7 were the sorted nuclei. R9 were determined to be Phox2b- by the isotype control.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Save the FACS program.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
(Optional). After FACS, combine 0.5 uL of the sorted sample in 9.5 uL Staining Buffer. Place 10 uL on a hemocytometer and view the nuclei under an epifluorescent microscope. Nuclei should be Hoescht+ and intact without large quantities of debris.a.	Note: we routinely avoided counting nuclei numbers using the hemocytometer since our sorted nuclei counts were so low (1000-10,000) that a hemocytometer did not give a good estimate of nuclei number. Instead, we typically provided the estimate from the FACS machine.
?. [Isolating nuclei for single nucleus RNA-seq, antibody staining and FACS]
Bring samples to core facility for 10x GEM generation and library preparation using the 10X Genomics platform.
?. [Preparing to isolate nuclei for RNA-Seq]
Prepare Lysis buffer:  ABCD1ChemicalStarting ConcentrationFinal ConcentrationAmount to add2Tris-HCL, pH 7.51 M10 mM400 uL3Sodium Chloride (NaCl)5 M10 mM80 uL4Magnesium Chloride1 M3 mM120 uL5Nonidet P40 Substitute10 %0.005%20 uL6RNase free water38.38 mL7Total volume40 mL
ABCD1ChemicalStarting ConcentrationFinal ConcentrationAmount to add2Tris-HCL, pH 7.51 M10 mM400 uL3Sodium Chloride (NaCl)5 M10 mM80 uL4Magnesium Chloride1 M3 mM120 uL5Nonidet P40 Substitute10 %0.005%20 uL6RNase free water38.38 mL7Total volume40 mL
?. [Preparing to isolate nuclei for RNA-Seq]
Prepare Staining buffer:  ABCD1ChemicalStarting ConcentrationFinal ConcentrationAmount to add2RNase free 10X PBS, pH 7.410X stock solution1X300  uL3Ultrapure 50 mg/mL BSA50 mg/mL1% w/v600 uL4Protector RNase inhibitor0.2 U/uL0.001 U/uL15 uL5Nuclease free water2085 uL6Total volume3000 uL
ABCD1ChemicalStarting ConcentrationFinal ConcentrationAmount to add2RNase free 10X PBS, pH 7.410X stock solution1X300  uL3Ultrapure 50 mg/mL BSA50 mg/mL1% w/v600 uL4Protector RNase inhibitor0.2 U/uL0.001 U/uL15 uL5Nuclease free water2085 uL6Total volume3000 uL
?. [Preparing to isolate nuclei for RNA-Seq]
Prepare Staining buffer:  ABCD1ChemicalStarting ConcentrationFinal ConcentrationAmount to add2RNase free 10X PBS, pH 7.410X stock solution1X990 uL3Ultrapure 50 mg/mL BSA50 mg/mL1% w/v1980 uL4Protector RNase inhibitor0.2 U/uL0.001 U/uL49.5 uL5Nuclease free water6880.5 uL6Total volume9900 uL
ABCD1ChemicalStarting ConcentrationFinal ConcentrationAmount to add2RNase free 10X PBS, pH 7.410X stock solution1X990 uL3Ultrapure 50 mg/mL BSA50 mg/mL1% w/v1980 uL4Protector RNase inhibitor0.2 U/uL0.001 U/uL49.5 uL5Nuclease free water6880.5 uL6Total volume9900 uL
?. [Preparing to isolate nuclei for RNA-Seq]
?. [Preparing to isolate nuclei for RNA-Seq]
Schedule FACS sort:Schedule FACS sort on MoFlo Astrios or an equivalent sorter with a 70 micron nozzle and the ability to detect DAPI/Hoescht.
?. [Preparing to isolate nuclei for RNA-Seq]
Schedule 10x Genomics Sequencing run:Schedule 10x Genomics Sequencing run in advance since samples are processed the day that nuclei are isolated.
?. [Dounce Homogenization to isolate single nuclei]
Get a large ice bucket.
?. [Dounce Homogenization to isolate single nuclei]
Prepare the appropriate quantities of Lysis buffer and Staining buffer (Steps 42 and 43). Place them on ice.
?. [Dounce Homogenization to isolate single nuclei]
Pre-cool the Dounce homogenizer and pestles on ice.
?. [Dounce Homogenization to isolate single nuclei]
Rinse large iridectomy scissors and Dumont #2 forceps with RNase Zap. Rinse with MilliQ water. Pre-cool large iridectomy scissors and Dumont #2 forceps on ice in an open 50 mL conical.
?. [Dounce Homogenization to isolate single nuclei]
Pre-cool large and small centrifuges to 4 C.
?. [Dounce Homogenization to isolate single nuclei]
Place a 6-well plate on ice. Fill wells with 6 mL of Lysis buffer.
?. [Dounce Homogenization to isolate single nuclei]
Get dry ice. Transfer sectioned human tissue to the bench on dry ice.
?. [Dounce Homogenization to isolate single nuclei]
Approximately 1 hour before FACS sort, put on appropriate PPE (gloves, lab coat, face mask, face shield).
?. [Dounce Homogenization to isolate single nuclei]
Use Dumont #2 forceps to transfer one Eppendorf tube with sectioned human tissue into 6 mL of Lysis buffer on 6 well plate.
?. [Dounce Homogenization to isolate single nuclei]
Chop rapidly with large iridectomy scissors for 1 minute.
?. [Dounce Homogenization to isolate single nuclei]
Using a 1000 uL pipet, transfer 2 mL of Lysis buffer + tissue/O.C.T. into a pre-cooled Dounce homogenizer.
?. [Dounce Homogenization to isolate single nuclei]
Homogenize tissue with 20 strokes of the loose pestle and 50 strokes of the tight pestle.Note: homogenization often induces suction and the formation of foam. Cover homogenizer with a paper towel and wear appropriate lab coat and PPE during this step to avoid getting splashed. Suction is particularly likely to occur for these tissue pieces because there are so many muscle fibers.
?. [Dounce Homogenization to isolate single nuclei]
Put the tissue through a MACS SmartStrainer (30 micron) resting atop a 50 mL conical.
?. [Dounce Homogenization to isolate single nuclei]
Chase with 2 mL of Lysis buffer to rinse the strainer. Switch out strainers if they appear to be getting clogged, between tissue samples, or after 3 uses.
?. [Dounce Homogenization to isolate single nuclei]
Repeat steps 54-59 until all Eppendorf tubes with tissue from the patient have been homogenized. Homogenized nuclei from the same patient may be combined into the same 50 mL conical.
?. [Dounce Homogenization to isolate single nuclei]
If nuclei from a second human subject are being run that day, repeat steps 51-60.Note: Since homogenization and FACS sorting takes a long time, we never ran nuclei from more than 2 human subjects in a single day to maximize RNA quality.
?. [Dounce Homogenization to isolate single nuclei]
Spin down Lysis buffer+nuclei+O.C.T. on large centrifuge at 590 RCG for 8 minutes at 4 degrees C.  Be sure to appropriately balance samples.
?. [Dounce Homogenization to isolate single nuclei]
After the centrifugation is over, use a Pipet-Aid to remove liquid from each 50 mL conical. Get the last bit of liquid out using a P1000.Note: you probably will not see a pellet at this stage. Be very, very careful not to pipet out the nuclei at the bottom of the 50 mL conical. I typically tilt the 50 mL conical to the side and leave a small quantity of liquid at the bottom.
?. [Dounce Homogenization to isolate single nuclei]
Resuspend nuclei from each patient in 990 mL staining buffer by pipetting 5-10 times with a P1000.
?. [Antibody staining nuclei prior to FACS]
Add 100 µL of Normal Donkey Serum for blocking.
?. [Antibody staining nuclei prior to FACS]
Add 1 µL of 2.5 mg/mL Hoechst.
?. [Antibody staining nuclei prior to FACS]
Leave on ice for 15 minutes to block.
?. [Antibody staining nuclei prior to FACS]
For each human tissue sample, label three Eppendorf tubes:a.	Isotype control tubeb.	Phox2b tube #1c.	Phox2b tube #2
?. [Antibody staining nuclei prior to FACS]
Transfer 330 µL of nuclei in blocking solution into each tube.
?. [Antibody staining nuclei prior to FACS]
Add 3.3 µL (3.3 µg) of goat IgG to the isotype control tube.
?. [Antibody staining nuclei prior to FACS]
Add 16.5 µL of PHOX2B antibody (3.3 µg) to Phox2b tubes #1 and #2.
?. [Antibody staining nuclei prior to FACS]
Attach Eppendorf tubes to Glas-Col Rotator with rubber bands. Rotate samples at speed 30 in the cold room on a rotator for 45 minutes.
?. [Antibody staining nuclei prior to FACS]
Add 1 mL of 1x PBS (rinse buffer) to each tube.
?. [Antibody staining nuclei prior to FACS]
Invert the tubes ~5 times to wash.
?. [Antibody staining nuclei prior to FACS]
Spin in an Eppendorf centrifuge for 5 minutes at 400 RCF at 4° C.
?. [Antibody staining nuclei prior to FACS]
While spinning, prepare 1,300 µL of 1:400 Alexa Fluor 647 Donkey anti-goat secondary antibody in staining buffer.
?. [Antibody staining nuclei prior to FACS]
When spin is over, pipette out supernatant without disturbing the pellet.
?. [Antibody staining nuclei prior to FACS]
Resuspend the pellet in 400 µL of secondary antibody in staining buffer.Note: pipette sample 5-10 times until any residual clumps are gone.
?. [Antibody staining nuclei prior to FACS]
Rotate samples at speed 30 in the cold room rotator for 30 minutes.a.	While waiting, label 1.5 mL Eppendorf tubes, 1 tube per patient. Add 5 uL staining buffer to the bottom of each tube. These are the collection tubes.b.	Also label Falcon round-bottom tubes, one tube per patient. These tubes will hold the sample to be sorted.c.	Place labeled 1.5 mL Eppendorf tubes and Falcon round-bottom tubes on ice to prepare for transport to FACS sorter.
?. [Antibody staining nuclei prior to FACS]
When 30 minutes is up, add 1 mL of 1x PBS (rinse buffer) to each tube with nuclei+secondary.
?. [Antibody staining nuclei prior to FACS]
Invert the tubes ~5 times to wash.
?. [Antibody staining nuclei prior to FACS]
Spin in an Eppendorf centrifuge for 5 minutes at 400 RCF at 4° C.
?. [Antibody staining nuclei prior to FACS]
Resuspend samples in 1 mL of staining buffer.Note: pipette sample 5-10 times until any residual clumps are gone. This is especially important to prevent clogging of the FlowMi strainer.
?. [Antibody staining nuclei prior to FACS]
Pipette the liquid into a P1000 pipette tip and attach the FlowMi strainer to the base. Strain through the FlowMi strainer into a spare Eppendorf tube.Note: if the FlowMi strainer clogs and liquid is not moving, do not panic! Slowly raise the plunger on your P1000 until it is fully raised. While holding the P1000 pipette tip, carefully remove the pipette tip from the P1000 pipettor. Then try to remove the FlowMi strainer from the base of the pipette tip with the lid of your Eppendorf while still holding the pipette tip securely. If you manage to do this successfully, pipette the liquid in the P1000 5 more times to thoroughly disrupt clumps, and try again with a new FlowMi strainer.
?. [Antibody staining nuclei prior to FACS]
Pipette the liquid into a new P1000 pipette tip. Strain through the top of the Falcon round-bottom tube by placing pipette tip flush against the strainer lid.
?. [Antibody staining nuclei prior to FACS]
Bring the samples and collection tubes to FACS on ice.
?. [FACS to isolate nuclei for RNA-seq]
Run isotype control sample first to determine appropriate gating.
?. [FACS to isolate nuclei for RNA-seq]
Sort Hoescht+, PHOX2B+ nuclei (with higher signal than isotype control) into the collection Eppendorf tubes with 5 uL staining buffer using 70 micron nozzle (this can sort up to 1,000 nuclei per microliter).Below is a sample image from a FACS sort. R7 were the sorted nuclei. R9 were determined to be PHOX2B negative by the isotype control.
?. [FACS to isolate nuclei for RNA-seq]
Save the FACS program for documenting how nuclei were selected for analysis.
?. [FACS to isolate nuclei for RNA-seq]
(Optional). After FACS, combine 0.5 uL of the sorted sample in 9.5 uL Staining Buffer. Place 10 uL on a hemocytometer and view the nuclei under an epifluorescent microscope. Nuclei should be Hoescht+ and intact without large quantities of debris.Note: we routinely avoided counting nuclei numbers using the hemocytometer since our sorted nuclei counts were so low (1000-10,000) that a hemocytometer did not give a good estimate of nuclei number. Instead, we typically provided the estimate from the FACS machine.
?. [FACS to isolate nuclei for RNA-seq]
Samples are ready for 10x GEM generation and library preparation using the 10X Genomics platform.