TITLE: Processing Human Colon "Myenteric Plexus" for Single Nuclei RNA-seq
AUTHORS: Christina M Wright,  Robert O Heuckeroth

[DESCRIPTION]
<div class = "text-blocks"><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.</div><div class = "text-block">OBJECTIVES: To obtain a suspension of single nuclei from human colon myenteric plexus that could be used for RNA-seq.</div><div class = "text-block">METHODS: 1) Human colon "myenteric plexus" is dissected from the bowel wall after labeling with a live cell dye and then frozen in OCT. 2) Frozen "myenteric plexus" is sectioned on a cryostat to generate small fragments and disrupt muscle and connective tissue. 3) RNA quality is assessed from a few sections using an Agilent Bioanalyzer. 4) Nuclei are obtained from frozen sections using Dounce homogenization and labeled with Hoechst 33342 dye. 5). Nuclei are separated from other cell debris by FACS. 6) RNA-seq data were obtained using the 10X Genomic single cell sequencing platform.</div><div class = "text-block">RESULTS: Live dye labeling works well on living tissue to visualize the ENS in human colon. Careful dissection is required to micro-dissect "myenteric plexus" from from within bowel wall muscle layers. A suspension of single nuclei is efficiently isolated by freezing isolated tissue in OCT, frozen sectioning, Dounce homogenizing, and FACS sorting Hoechst 33342 stained nuclei. The isolated nuclei are suitable for sequencing on the 10X Genomics single cell sequencing platform. This procedure dramatically enriched for cells in the region of the myenteric plexus, but unfortunately only a small percentage of the cells isolated are myenteric neurons. In addition to myenteric neurons, we obtained data from enteric glia, smooth muscle, muscularis macrophage, interstitial cells of Cajal, and PDGFRalpha+ cells that are known to be closely associated with myenteric plexus.</div><div class = "text-block">CONCLUSION:  We tried many approaches to optimize this procedure including additional steps to enrich for myenteric neuron nuclei. These additional procedures invariably reduced total cell yield without enriching significantly for neuronal nuclei. We do not know yet why neuronal nuclei remain a small component of our total nuclei population, but suspect that neuronal nuclei are more fragile than other nuclei isolated. This approach generated valuable data at a single nucleus RNA-seq level for all cell types within the muscle wall known to control bowel motility.</div></div>

[STEPS]
?. [PREPARING SYLGARD DISH FOR PINNING TISSUE]
Prepare Sylgard by combining curing agent with polymer as described in the kit.
?. [PREPARING SYLGARD DISH FOR PINNING TISSUE]
Carefully drip Sylgard into a 35x10 mm dish until the dish is halfway full.
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Allow to cure in a fume hood for at least 3 days.
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After curing is complete, superglue the 35x10 mm dish to the center of a Fisherbrand Petri dish with clear lid. The Petri dish will provide containment for leakage from the 35x10 mm dish, and also hold ice chips to surround the tissue with ice.
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Insert Minutien pins (>10) into the 35x10 mm dish to prepare it for pinning human tissue.Krebs-Ringer Solution
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Combine the NaCl, KCl, CaCl2, MgSO4, NaH2PO4, D-Glucose (see RECIPE below for amounts).
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Add MilliQ water to 990 mL. Stir continuously.
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Adjust the pH to 7.4 using 4 N HCl.
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Store at 4 oC.
?. [PREPARING 4-DI-2-ASP]
Weigh 9.8 mg of 4-Di-2-Asp powder in a 50 mL conical.
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Combine 4-Di-2-ASP with 50 mL sterile dPBS to make 50 mL of 500 uM 4-Di-2-ASP stock solution. This can be stored at room temperature for extended periods. (We do not know how long it will be effective)
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Half hour before the tissue arrives:     a.	Place chilling block in the -20 oC freezer     b.	Place ~20 mL of Krebs-Ringer into a 50 mL conical. Bubble 5% CO2, 95% O2 through the Ringer using a 10 mL serological pipette.  This is for Room temperature, oxygenated Krebs-Ringer     c.	Also keep at least 50 mL Krebs-Ringers solution at 4 °C (you do not need to oxygenate this). This is for Cold Krebs-Ringer     d.	Spray off forceps and scissors with RNase Away, and thoroughly rinse/soak in water     e.	Fetch dry ice.     f.	        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.
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Transfer tissue, on ice, from the hospital’s Pathology unit to your laboratory in sterile 1x PBS in a 50 mL conical or similar container. (~30 minutes)
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Upon arrival to lab, don appropriate personal protective equipment  (PPE) = (gloves, lab coat, face mask, face shield,).
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Place tissue into cold Krebs-Ringer in a large sterile Petri dish.
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Trim off surrounding fat using Dumont #2 forceps and a scissors.
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Cut a ~1 cm x 1cm piece of tissue. Remove as much epithelium and submucosa as possible with scissors.
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Transfer the smaller tissue piece (mostly muscularis) into the 35x10 mm dish with pins. Leave the rest of the human tissue on a Petri dish on ice.
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Add 2.7 mL of room temperature, oxygenated Krebs-Ringer to the small tissue piece in the 35x10 mm dish.	Note: it is important that the Krebs-Ringer solution be at room temperature for this part; otherwise, the 4-Di-2-ASP will not be absorbed by myenteric plexus cells.
?. [HUMAN COLON TISSUE HANDLING TO ISOLATE "MYENTERIC PLEXUS"  ON DAY OF TISSUE COLLECTION]
Carefully pin the tissue flat, stretching it to maximum possible capacity.Note: The more the tissue is stretched, the better the 4-Di-2-ASP will penetrate.
?. [HUMAN COLON TISSUE HANDLING TO ISOLATE "MYENTERIC PLEXUS"  ON DAY OF TISSUE COLLECTION]
Add 300 uL of 500 uM 4-Di-2-ASP stock solution to the Krebs-Ringer solution already in the dish. Pipette up and down until the 4-Di-2-ASP has spread evenly across the dish.
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Set timer for 15 minutes.
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During this time, prepare two Biopsy cryomolds by labeling them with the sample ID number. Label one ‘plexus’ and the second one ‘muscle’. Fill cryomolds about halfway with O.C.T. and place them on ice.
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After 15 minutes, remove the Krebs-Ringer with 4-Di-2-ASP from the dish with colon tissue.
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Replace with 2 mL cold Krebs-Ringer solution to cover human colon tissue.
?. [HUMAN COLON TISSUE HANDLING TO ISOLATE "MYENTERIC PLEXUS"  ON DAY OF TISSUE COLLECTION]
Add ice to larger petri dish surrounding the 35x10 mm dish.
?. [HUMAN COLON TISSUE HANDLING TO ISOLATE "MYENTERIC PLEXUS"  ON DAY OF TISSUE COLLECTION]
Remove chilled block from the -20 oC and place it on the stage to the fluorescent dissecting microscope. Place petri dish with tissue on top of the chilled block. Placed Biopsy cryomold labeled ‘Plexus’ in the ice on the larger surrounding petri dish.
?. [HUMAN COLON TISSUE HANDLING TO ISOLATE "MYENTERIC PLEXUS"  ON DAY OF TISSUE COLLECTION]
Check that the myenteric plexus is labeled by viewing the tissue in the 488 nm channel on the fluorescent dissecting microscope.
?. [HUMAN COLON TISSUE HANDLING TO ISOLATE "MYENTERIC PLEXUS"  ON DAY OF TISSUE COLLECTION]
With #5 Dumont forceps, carefully dissect away the muscle, exposing the myenteric plexus beneath.Note: move very slowly whenever you are working with sharp forceps and human tissue. NEVER point the tip of the forceps toward your fingers or hands. If you accidently stab yourself, immediately rinse the wound in the sink and follow the appropriate safety protocols at your institution.
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Carefully separate myenteric plexus from surrounding tissue. Place isolated pieces of plexus in cold OCT in the Biopsy cryomold.Note: separation will never be perfect. There will always be muscle and other cells dissected out along with the plexus. The goal is just to enrich for plexus not isolate it entirely.
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When as much plexus has been separated as possible for this tissue piece, place the cryomold containing the cold OCT+ plexus in the 2-Methylbutane in the fume hood.
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Cut a piece of the remaining muscle. Place this in the second cryomold labeled ‘Muscle’. Place this in the 2-Methylbutane in the fume hood.Note: this additional step is done purely for the purposes of assessing RNA quality in the tissue.
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Wait 5 minutes for it to freeze. Then transfer to a -80 oC freezer in appropriate containment (e.g. plastic bags in labeled boxes).
?. [HUMAN COLON TISSUE HANDLING TO ISOLATE "MYENTERIC PLEXUS"  ON DAY OF TISSUE COLLECTION]
If there is more human tissue, repeat steps 6-22. The maximum we process is typically 3 pieces per sample since each piece takes ~30-45 minutes.
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Clean plates with RNase away and bleachNote: all plates should be handled extremely carefully, particularly the pins since there is the potential for human pathogen exposure.
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Autoclave tools.
?. [PREPARING KREBS-RINGER SOLUTION]
RECIPE for KREBS-RINGER SOLUTIONChemical	                                                      Final concentration          Molecular weight	                        For 1 LSodium chloride (NaCl)	                                    118 mM	                        58.44 g/mol	                                6.896gPotassium chloride (KCl)	                                        4.6 mM	                        74.55g/mol	                                0.343gCalcium chloride dihydrate (CaCl2 · 2H2O)	        2.5 mM	                      147.01g/mol	                                0.368gMagnesium sulfate anhydrous (MgSO4)	                1.2 mM	                      120.37g/mol	                                0.1444gSodium phosphate monobasic (NaH2PO4)	        1 mM	                      119.98g/mol	                                0.120gD-(+)-Glucose	                                                      11 mM	                        84.01g/mol	                                2.100gSodium bicarbonate (NaHCO3)	                      25 mM	                      180.16g/mol	                                1.982g
?. [PREPARING KREBS-RINGER SOLUTION]
Add NaHCO3.
?. [HUMAN COLON SECTIONING IN PREPARATION FOR ISOLATION OF NUCLEI FOR RNA-SEQ]
Fill an ice bucket with dry ice.
?. [HUMAN COLON SECTIONING IN PREPARATION FOR ISOLATION OF NUCLEI FOR RNA-SEQ]
RNaseZAP a Dumont #2 Forceps. Rinse with deionized MilliQ water.
?. [HUMAN COLON SECTIONING IN PREPARATION FOR ISOLATION OF NUCLEI FOR RNA-SEQ]
Transfer tissue labeled “Plexus” in biopsy cryomold on dry ice to Cryostat set to -20 °C.
?. [HUMAN COLON SECTIONING IN PREPARATION FOR ISOLATION OF NUCLEI FOR RNA-SEQ]
Freeze the tissue onto a metal cryostat mounting device using a minimum of O.C.T. compound.
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While waiting for tissue to freeze, label 3 DNA LoBind Tubes: “Plexus”, “ Plexus”, and “ Plexus RNA”. Place these on dry ice or in the 20 oC for at least 3 minutes before sectioning so they have time to cool.
?. [HUMAN COLON SECTIONING IN PREPARATION FOR ISOLATION OF NUCLEI FOR RNA-SEQ]
Place Dumont #2 Forceps in cryostat to chill.
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Mount the tissue, insert the blade, and begin making 100 micron sections.
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Using the Dumont #2 Forceps, collect all sections in “ Plexus” tubes EXCEPT 2-5 sections. Put 2-5 random sections in tube labeled “ Plexus RNA”.
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Return the tubes with tissue to the -80 oC on dry ice.
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Section through the Muscle piece until the middle of the tissue is reached. Collect 2-5 sections in an Eppendorf tube. Label this Eppendorf tube “ Muscle RNA”
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Return tube to the -80 oC on dry ice.
?. [EXTRACTING RNA FOR BIOANALYZER SUBMISSION TO ASSESS RNA QUALITY BEFORE RNA-SEQ]
Follow the instructions on the Qiagen RNeasy Micro Kit as detailed below:
?. [EXTRACTING RNA FOR BIOANALYZER SUBMISSION TO ASSESS RNA QUALITY BEFORE RNA-SEQ]
Remove tubes labeled “RNA” from the -80 oC. There should be one each for plexus and for surrounding muscle.
?. [EXTRACTING RNA FOR BIOANALYZER SUBMISSION TO ASSESS RNA QUALITY BEFORE RNA-SEQ]
Add 350 uL of buffer RLT plus to each tube.
?. [EXTRACTING RNA FOR BIOANALYZER SUBMISSION TO ASSESS RNA QUALITY BEFORE RNA-SEQ]
Homogenize the lysate by vortexing for 30 seconds.
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Run through gDNA spin column. Centrifuge for 30 seconds at >= 8000 g.
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Discard the column and keep the flow-through.
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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 SUBMISSION TO ASSESS RNA QUALITY BEFORE RNA-SEQ]
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 15 seconds at > 8,000 RCF. Discard the flow thru.
?. [EXTRACTING RNA FOR BIOANALYZER SUBMISSION TO ASSESS RNA QUALITY BEFORE RNA-SEQ]
Add 700 uL of buffer RW1 to the RNEasy MinElute spin column. Close the lid and centrifuge for 15 seconds at 8,000 RCF. Discard the flow-through.
?. [EXTRACTING RNA FOR BIOANALYZER SUBMISSION TO ASSESS RNA QUALITY BEFORE RNA-SEQ]
Add 500 uL Buffer RPE to the RNeasy MinElute spin column. Close lid gently, and centrifuge for 15 seconds at > 8000 RCF to wash the spin column membrane.
?. [EXTRACTING RNA FOR BIOANALYZER SUBMISSION TO ASSESS RNA QUALITY BEFORE RNA-SEQ]
Add 500 uL of 80% ethanol to the RNEasy MinElute column. Close the lid and centrifuge for 2 minutes at 8,000 RCF to wash the spin column membrane. Discard the collection tube with the flow-through.
?. [EXTRACTING RNA FOR BIOANALYZER SUBMISSION TO ASSESS RNA QUALITY BEFORE RNA-SEQ]
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 SUBMISSION TO ASSESS RNA QUALITY BEFORE RNA-SEQ]
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.
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Run on the Agilent RNA 2100 Bioanalyzer with the Agilent RNA 6000 Pico Kit, along with any additional samples.
?. [PREPARATION FOR ISOLATING NUCLEI FOR SINGLE NUCLEUS RNA-SEQ]
Prepare Hoechst dye 2.5 mg/mL:Dilute 10 mg/mL Hoescht 1:4 in nuclease-free waterUsing 3 mL syringe and 0.22 micron filter, filter the Hoescht.Store at 4 degrees C.
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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.
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Autoclaving:-Rinse large iridectomy scissors and Dumont #2 forceps with RNaseZAP followed by MilliQ water.-Place in small autoclave bag.-Autoclave before use.
?. [PREPARATION FOR ISOLATING NUCLEI FOR SINGLE NUCLEUS 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.Note: Nuclei are isolated by FACS on the day of 10X Genomics sequencing analysis
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Schedule 10x Genomics Sequencing runNote: Nuclei are isolated by FACS on the day of 10X Genomics sequencing analysis
?. [ISOLATING NUCLEI FOR SINGLE NUCLEUS RNA-SEQ]
Get a large ice bucket.
?. [ISOLATING NUCLEI FOR SINGLE NUCLEUS RNA-SEQ]
Prepare the appropriate quantities of Lysis buffer and Staining buffer (see steps 66 and 67). Place them on ice.
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Precool the Dounce homogenizer and pestles on ice.
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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.
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Pre-cool large centrifuge to 4 oC.
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Place a 6-well plate on ice. Fill wells with 6 mL of Lysis buffer (Step 66).
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Get dry ice. Transfer sectioned human tissue to the lab bench on dry ice.
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Approximately 3 hour before FACS sort, put on appropriate Personal Protective Equipment (PPE) (gloves, lab coat, face mask, face shield) to protect from human pathogen exposure.
?. [ISOLATING NUCLEI FOR SINGLE NUCLEUS RNA-SEQ]
Use Dumont #2 forceps to transfer one Eppendorf tube with sectioned myenteric human tissue into 6 mL of Lysis buffer on 6 well plate.
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Chop rapidly with large iridectomy scissors for 1 minute.
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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]
Homogenize tissue with 15 strokes of the loose pestle and 40 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.
?. [ISOLATING NUCLEI FOR SINGLE NUCLEUS RNA-SEQ]
Put the tissue through a MACS SmartStrainer (30 micron) resting atop a 50 mL conical.
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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.
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Repeat steps 79-84 until all Eppendorf tubes with tissue from a single human subject have been homogenized. Homogenized nuclei from the same human subject may be combined into the same 50 mL conical.
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If nuclei from a second human subject colon are being run that day, repeat steps 76-85.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]
Spin down Lysis buffer+nuclei+OCT on large centrifuge at 590 RCF for 8 minutes at 4 oC.  Be sure to appropriately balance samples.
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While tissue is spinning, label 1.5 mL Eppendorf tubes, 1 tube per human subject. Add 5 uL Staining buffer (Step 67) to the bottom of each tube. These are the collection tubes.
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Also label Falcon round-bottom tubes, one tube per human subject. These tubes will hold the sample to be sorted.
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Place labeled 1.5 mL Eppendorf tubes and Falcon round-bottom tubes on ice to prepare for transport to FACS sorter.
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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.
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Resuspend in 1 mL Staining buffer by pipetting 5-10 times with a P1000.Note: the FlowMi strainer may clog if there are clumps in the sample. Be sure to pipette and disperse clumps thoroughly.
?. [ISOLATING NUCLEI FOR SINGLE NUCLEUS RNA-SEQ]
Add 1 uL of 2.5 mg/mL Hoechst.
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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.
?. [ISOLATING NUCLEI FOR SINGLE NUCLEUS RNA-SEQ]
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.
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Bring the samples and collection tubes to the FACS on ice.
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Sort Hoescht+ nuclei into the collection Eppendorf tubes with 5 uL staining buffer using 70 micron nozzle (this can sort up to 1,000 nuclei per microliter).We have found success with the gating scheme below which removes debris and isolates nuclei:
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Save the FACS program to document what you did during nucleus isolation.
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(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.
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Isoled nuclei are ready for 10x GEM generation and library preparation using 10X Genomics protocols.
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Prepare Lysis buffer:  ABCD1ChemicalStarting ConcentrationFinal ConcentrationAmount to add2Tris-HCl pH 7,51 M10 mM400 uL3Sodium Chloride (NaCl)5 M10 mM80 uL4Magnesium Chloride (MgCl2)1 M3 mM120 uL5Nonidet P4010%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 Chloride (MgCl2)1 M3 mM120 uL5Nonidet P4010%0.005%20 uL6RNase free water38.38 mL7Total volume40 mL
?. [PREPARATION FOR ISOLATING NUCLEI FOR SINGLE NUCLEUS 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