TITLE: Single Nucleus Drop-seq (snDrop-seq)
AUTHORS: Song Chen, Blue B. Lake, Sarah Urata, Kun Zhang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol presented here is a Drop-Seq protocol modified for single nuclei. </div><div class = "text-block">The original Drop-Seq protocol comes from the McCarroll Lab in the Department of Genetics, Harvard Medical School.</div><div class = "text-block"><a href="http://mccarrolllab.org/download/905/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://mccarrolllab.org/download/905/</span></a></div></div>

[STEPS]
?. [Droplet Formation]
Prepare Pump SystemPower on the syringe pumps (switches located on the back left of the pump)Set up correct flow rates using the screen on the pumpOil:       15,000 ul/hrCells:     4,000 ul/hrBeads:   4,000 ul/hr
?. [Droplet Formation]
Prepare microfluidic cellCut and remove protective plastic top layer for one device on the chipCut the outflow tubing to proper length and connect it to the device so that the collection from the outflow can be safely collectedUse clean 15 mL tubes to catch the dropletsUse a waste container to catch waste
?. [Droplet Formation]
Prepare the beadsTake an aliquot of beads (final concentration: 120,000 beads/mL)Spin down in a tabletop centrifugeRemove the TE-TW storage bufferWash with DTT minus lysis bufferResuspend in Lysis buffer
?. [Prepare Buffers and Solutions]
Cell Loading Buffer (makes)Prepare enough for the number of samples () that will be processed in one experiment
1 ml
[per sample]
[20% Ficoll PM-400]
[1X PBS]
?. [Prepare Buffers and Solutions]
Lysis Buffer (makes)Prepare enough for the number of samples () that will be processed in one experimentAdd this just prior to starting each DropSeq experiment
1.2 ml
[per sample]
[H2O]
[20% Sarkosyl]
[0.5 M EDTA]
[2M Tris, pH 7.5]
[1M DTT]
?. [Prepare Buffers and Solutions]
BSAMake a 10% stock solution using BSA powder
?. [Prepare Buffers and Solutions]
PBS-BSA: make this fresh before each experimentCell Loading Buffer0.01% BSA (use the 10% stock)
?. [Prepare Buffers and Solutions]
6X SSCMake 6X SSC working stock from 20X SSC
?. [Prepare Buffers and Solutions]
TE-SDS (make)10 mM Tris pH 8.0 + 1 mM EDTA0.5% SDS
50 ml
?. [Prepare Buffers and Solutions]
TE-TW (make)10 mM Tris pH 8.0 + 1 mM EDTA0.01% Tween-20
500 ml
?. [Prepare Buffers and Solutions]
10 mM Tris pH 8.0Make 10 mM Tris pH 8.0 working stock from 2M Tris pH 8.0
?. [Droplet Formation]
Arrangement of components
?. [Droplet Formation]
Preparing the Barcoded Beads from Dry ResinThe beads will arrive as a dry resin. To prepare the beads for an experiment from the dry resin, follow these steps:Wash the resin with Wash twice with Resuspend in Pass through 100 micron strainerCount the beads using a Fuchs-Rosenthal hemocyctometerStore the counted beads at
[ethanol]
[TE-TW]
[TE-TW]
4 °C
To pellet the beads when washing, centrifuge at 1000xg for
Do NOT vortex the barcoded beads. Vortexing will shear the beads and cause damage. To mix, simply invert tube gently a couple of times until mixed.
?. [Droplet Formation]
Prepare nuclei according to the protocol "Isolation of single nuclei from solid tissues" steps 1-14. dx.doi.org/10.17504/protocols.io.ufketkwDilute nuclei to 100 cells/ul. Use PBS-BSA for final dilution
?. [Droplet Formation]
Prepare syringes and lines for microfluidic devicePrepare enough syringes and lines for all samples at once (4-6 samples per experiment)Oil syringe: 10 mlCell/Sample syringes: 3 mlBead syringes: 3 ml with magnetic mixing discMeasure out tubing for the lines; make sure they are long enough to reach the microfluidic device from where the syringes will sit on the pumpsFor each line cut one blunt end (for blunt end needle) and one angled end (insert into the device)UV all tubing and needles
?. [Droplet Formation]
Load SyringesFlush the tubing with corresponding buffer; keep syringes vertical when flushingUse  to flush syringe/tube for each sampleUse  to flush syring/tube for each bead setupLoad the syringes; keep syringes vertical when loadingLoad cells/nuclei into cell syringes, making sure there are no air bubblesLoad beads into bead syringes, making sure there are no air bubblesAfter beads are loaded in the syringe, keep horizontal to prevent cloggingUse to flush syringe/tube for each sampleUse to flush syring/tube for each bead setupLoad the syringes; keep syringes vertical when loadingLoad cells/nuclei into cell syringes, making sure there are no air bubblesLoad beads into bead syringes, making sure there are no air bubblesAfter beads are loaded in the syringe, keep horizontal to prevent clogging
To load syringe, firmly press the tip of a 1000 ul pipette into the head of the syringe and slowly pull back on the plunger to draw in the solution. Pressing the tip in firmly helps reduce the introduction of bubbles. While holding the syringe in a vertical orientation, gently push out the air bubbles. Affix a 26G needle and tubing.
Before droplet generation, coat connecting tubing and syringes with 1% BSA to prevent non-specific binding of nuclei to the surface, and then rinse with PBS
[cell buffer]
[ DTT minus lysis buffer]
?. [Droplet Formation]
System AssemblyPlace oil syringe in pump and secure; using the screen, hold the free tube over the waste collection, then slowly move the pusher to the plunger until oil drips outPush the angled end of the tubing into the devicePlace cell/sample syringe in pump and secure; using the screen, hold the free tube over the wase collection, then slowly move the pusher to the plunger until buffer drips outPush the angled end of the tubing into the deviceBefore placing the bead syringe, turn on the magnetic mixer for stirring disc (speed 25-30). Carefully rotate bead syringe by hand to mix up the beads in the lysis buffer. Place bead syringe in pump and secure. Make sure the magnetic disc is circling up and down vertically in the syringe to keep the beads well mixed.Using the screen, hold the free tube over the wase collection, then slowly move the pusher to the plunger until buffer drips outPush the angled end of the tubing into the device
?. [Droplet Formation]
Run Pumps for Droplet FormationStart oil pumpStart bead pumpStart cells/sample pump
?. [Droplet Formation]
Check for Droplet FormationHave outflow line go to wasteWatch the beads move from the syringe to the deviceOnce the beads get to the bead chamber in the device, use a clean microscope slide to catch a drop of outputCheck slide under a different microscope to see if droplets are uniform in size and shapeOnce droplets are uniform begin collecting
?. [Droplet Formation]
CollectCollect uniform droplets in 15 ml Falcon tubes until cells/sample runs outThe pump will sound an alarm once the cell/sample runs out; turn off alarmSTOP collecting in the 15 ml Falcon tube immediately (place a waste container under the outflow)
?. [Droplet Formation]
Stop pumpsStop all pumps in any orderPlace the 15 ml Falcon tube with droplets on ice
?. [Droplet Formation]
When running more than one sample Repeat Step 20- Step 24 for each sample
?. [Droplet Formation]
Clean up (AFTER Reverse Transcription is set up to incubate)Turn off magnetic mixerRemove syringes from pumps; remove tubing from microfluidic devicePower off pumps (switches located on the back left of the pump)Cell/sample outflow tubing goes in biohazardous solid wasteAll other lines can go in regular wasteCell/sample syringes go in biohazardous solid wasteAll other syringes can go in regular wasteCell/sample needles go in biohazardous sharps wasteAll other needles go in regular sharps waste
?. [STAMP Collection]
Single cell/nuclei Transcriptomes Attached to MicroParticles (STAMP)Prepare waste and collection tubeNeed one 50 mL Falcon tube for waste (can use same waste from preparing syringes)Need one new clean 50 mL Falcon tube for collectionNeed one 30 um UberstrainerAutopipette, 5 mL pipette, and attachment for strainer
STAMPs are the barcoded beads with mRNA attached; for stability, work on ice/keep cold when working with STAMPs/RNA
Add Maxima H- RTase to RT Mix (Step 10)
?. [STAMP Collection]
BreakageBe careful not to disturb the oil/droplet band (direct buffer down the side of the 15 mL Falcon tube and control output so there is smooth flow)Before droplet breakage, add  and incubate in water bath at  for Let sit on ice for  Remove the oil from the bottom of the 15 mL Falcon tubeAdd  Add  directly to the oil/droplet band to break the droplets; dispense in a circular motion directly over/onto the droplet badGently roll/rotate the 15 mL Falcon tube on ice to help break the dropletsBefore droplet breakage, add and incubate in water bath at forLet sit on ice for Remove the oil from the bottom of the 15 mL Falcon tubeAdd  Add  directly to the oil/droplet band to break the droplets; dispense in a circular motion directly over/onto the droplet badGently roll/rotate the 15 mL Falcon tube on ice to help break the dropletsAdd Add directly to the oil/droplet band to break the droplets; dispense in a circular motion directly over/onto the droplet bad
[mineral oil]
72 °C
[cold 6X SSC]
[Perfluorooctanol (PFO)]
?. [STAMP Collection]
Filter/CollectionSet up the 30 um Uberstrainer over the waste tubePlace strainer over waste tube (goal: save beads, discard supernatant)Attach autopipette attachment to strainerAngle/tilt tube so that no liquid will go up through the autopipette when using it for a vacuumUse  to moisten the filterFilter the beads (over waste tube)Pass the aqueous (top layer) phase through the strainerPass the organic (bottom layer) phase through the strainerUse a couple mililiters cold 6X SSC to rinse the 15 mL Falcon tube to try and get all the beads from the sides; pass the rinse through the strainerRinse strainer/filter twice with cold 6X SSCCollect the beads (over new/clean 50 mL Falcon tube)Carefully reverse the strainer over a new/clean 50 mL Falcon tubeWash strainer with  . Repeat four more times (5 washes total)Visually inspect the strainer to make sure all the beads have come off itUse to moisten the filterFilter the beads (over waste tube)Pass the aqueous (top layer) phase through the strainerPass the organic (bottom layer) phase through the strainerUse a couple mililiters cold 6X SSC to rinse the 15 mL Falcon tube to try and get all the beads from the sides; pass the rinse through the strainerRinse strainer/filter twice with cold 6X SSCCollect the beads (over new/clean 50 mL Falcon tube)Carefully reverse the strainer over a new/clean 50 mL Falcon tubeWash strainer with  . Repeat four more times (5 washes total)Visually inspect the strainer to make sure all the beads have come off itCollect the beads (over new/clean 50 mL Falcon tube)Carefully reverse the strainer over a new/clean 50 mL Falcon tubeWash strainer with  . Repeat four more times (5 washes total)Visually inspect the strainer to make sure all the beads have come off itWash strainer with . Repeat four more times (5 washes total)
[cold 6X SSC]
[cold 6X SSC]
?. [STAMP Collection]
Spin DownUsing a , spin down the 50 mL Falcon tube at 1000xg for Carefully remove, supernatant, leaving approximately
[centrifuge]
[cold 6X SSC]
?. [STAMP Collection]
Transfer to 1.5 mL tubeResuspend beads in remaining Transfer to a new/clean 1.5 mL microfuge tubeSpin down (1000xg for  )Wash twice with  Wash with  Remove as much of the 5X RT buffer as possible without taking up any beadsSpin down (1000xg for )Wash twice with Wash with
[cold 6X SSC]
[cold 6X SSC]
[5X RT buffer]
?. [Reverse Transcription]
This step generates cDNA strands on the RNA hybridized to the bead primers. One RT mix is sufficient for processing approximately 90,000 beads.Add to the beadsIncubate at for Incubate at for Wash beads once with Wash beads twice with If proceeding to Exonuclease I treatment, wash once with
[RT Mix]
[with rotation]
[with rotation]
[TE-SDS]
[TE-TW]
[10 mM Tris pH 8.0]
Stopping point: if stopping, stop at TE-TW wash step. Beads can be stored at
?. [Prepare Buffers and Solutions]
RT mix (makes)Add after beginning breakage portion of the protocol (Step 27).
200 µl
[H2O]
[Maxima 5x RT Buffer]
[20% Ficoll PM-400]
[10 mM dNTPs]
[RNase Inhibitor]
[50 uM Template Switch Oligo]
[Maxima H- RTase]
?. [Exonuclease I Treatment]
This step chews back the excess bead primers that did not capture an RNA molecule. One Exonuclease Mix is sufficient for processing approximately 90,000 beads.After washing once with , resuspend in Incubate at for Wash beads once with Wash beads twice with If proceeding to PCR, wash once with
[10 mM Tris pH 8.0]
[Exonuclease Mix]
[with rotation]
[TE-SDS]
[TE-TW]
[H2O]
Stopping point: if stopping, stop at TE-TW wash step. Beads can be stored at
?. [Prepare Buffers and Solutions]
Exonuclease Mix (makes)
200 µl
[10x Exo I Buffer]
[H2O]
[Exo I]
?. [PCR]
This steps utilizes PCR to amplify the cDNA constructed during the Reverse Transcription step.Bead CountAfter washing once with , spin down to pellet beadsRemove supernatantAdd  Mix well by pipette to evenly resuspend the beadsQuickly remove   and pipette into a Fuchs-Rosenthal hemocytometer chamberCount all 16 boxesAdd Mix well by pipette to evenly resuspend the beadsQuickly remove   and pipette into a Fuchs-Rosenthal hemocytometer chamberCount all 16 boxesQuickly remove  and pipette into a Fuchs-Rosenthal hemocytometer chamber
[H2O]
[H2O]
20 µl
?. [PCR]
Calculate number of beads to split into PCR tubesCalcuate the concentraion (beads/ul) = (#beads counts/16) x 5Apportion 2,000 beads into each PCR tube (want approximately 100 STAMPs per PCR tube)
?. [PCR]
Prepare Master MixMaster Mix ()
[per PCR reaction]
[H2O]
[100 uM SMART PCR Primer]
[2x Kapa HiFi Hotstart ReadyMix]
?. [PCR]
PCRSpin down tubesAdd   to each reactionMix well by pipetteRun the following program on a thermocyclerAdd  to each reactionMix well by pipetteRun the following program on a thermocycler for  4 cycles of for  for  for 13 cycles of for  for  for Then for
[PCR Master Mix]
95 °C
98 °C
65 °C
72 °C
98 °C
67 °C
72 °C
72 °C
[forever]
?. [Purification and Quantification]
Magnetic Bead PurificationUsing either KAPA PureBeads or AMPure XP beads, add  to each PCR sample (this is a 0.6x beads to samples ratio based on volume)Elute in
[room temperature magnetic beads]
[H2O]
?. [Purification and Quantification]
QuantificationCan be performed by preferred method (qPCR, Qubit Assay, BioAnalyzer, Tapestation); we used Qubit Assay.Use  for this quantification
[sample input]
The yield for 2000 beads generated from a 50 cell/ul total yield should be approximately 1-100ng.QC Cutoff: > 1ng total
?. [Purification and Quantification]
Amplified cDNA GelConfirm average size of cDNA library via gel electrophoresisAverage size should be between 1300-2000 bp
?. [Tagmentation]
This step simultaneously tags and fragments the cDNA library using transposomes
?. [Tagmentation]
Nextera KitPreheat a thermocycler to Working in a cold rack/on ice: for each sample, combine  with H2O in a total volume of To each tube, add  and  (the total volume of the reaction is now )Mix by pipette approximately 5 timesSpin downIncubate at   for  Add   (the total volume of the reaction is now  )Mix by pipette approximately 5 timesSpin down (bubbles are normal)Incubate at  for Add  (the total volume of the reaction is now )Mix by pipette approximately 5 timesSpin down (bubbles are normal)Incubate at  for
55 °C
[purified cDNA]
5 µl
[Nextera TD buffer "Tagment DNA"]
[Amplicon Tagment enzyme "Amplicon Mix"]
20 µl
55 °C
[Neutralization Buffer]
25 µl
0 Room temperature
?. [Tagmentation]
PCR work up of tagmented libraryPrepare PCR Master Mix ()
[per PCR reaction]
[Nextera PCR mix]
[H2O]
[10 uM New-P5-SMART PCR hybrid oligo]
?. [Tagmentation]
PCRAdd  to each tubeTo each individual tube, add  The total volume of the reaction is now  for 12 cycles of for  for  for Then for
[Tagmentation PCR Master Mix]
[10 uM Nextera N7XX oligo]
50 µl
95 °C
95 °C
55 °C
72 °C
72 °C
[forever]
?. [Purification and Quantification]
Magnetic Bead PurificationUsing either KAPA PureBeads or AMPure XP beads, add  to each PCR sample (this is a 0.6x beads to samples ratio based on volume)Elute in  to
[room temperature magnetic beads]
12 µl
[H2O]
?. [Purification and Quantification]
QuantificationCan be performed by preferred method (qPCR, Qubit Assay, BioAnalyzer, Tapestation); we used Qubit Assay.Use  for this quantificationQuantify according to manufacturer's instructionsConvert ng/ul to nM
[sample input]
The yield for 2000 beads generated from a 50 cell/ul final cell concentration should be approximately 400-1000 pg/ul.QC Cutoff >1ng
?. [Purification and Quantification]
Sequencing Library GelConfirm average size of Sequencing library via gel electrophoresisAverage size should be between 500-680 bp
Smaller-sized libraries will have more polyA reads; larger libraries may have lower sequence cluster density and cluster quality. Although the target size is 500-680 bp, the range can be as broad as 420-700 bp.
?. [Purification and Quantification]
2nd Magnetic Bead PurificationUsing either KAPA PureBeads or AMPure XP beads, perform a 0.6x purificationPurify according to manufacturer's instructionsElute in  to
12 µl
[H2O]
?. [Sequencing]
MiSeq Sequencing  - QC for estimation of library quality and number of nuclei capturedPool, denature, and dilute to loading concentration according to manufacturer's instructionsSequencing specificationsRead 1: 30 bpRead 2: 75~100 bpRead 1 Index: 8 bpCustom Read 1 primerHiseq 2500 Sequencing1. Combine 8-12 snDrop-seq libraries to make a 10 µl library pool at 3 nM for denaturation. 2. After final dilution, load a combined library at 12 pM to the sequencer Read 1: 30 bpRead 2: 75~100 bpRead 1 Index: 8 bpCustom Read 1 primerHiseq 2500 Sequencing1. Combine 8-12 snDrop-seq libraries to make a 10 µl library pool at 3 nM for denaturation. 2. After final dilution, load a combined library at 12 pM to the sequencer Read 1: 30 bpRead 2: 75~100 bpRead 1 Index: 8 bpCustom Read 1 primer
?. [Prepare Buffers and Solutions]
1X PBSMake a 1X stock solution using by diluting 10X PBS into nuclease-free distilled water
?. [Sequencing]
snDrop-seq data processingMapping, demultiplexing and QC processing: https://github.com/chensong611/Dropseq_pipelinePaired-end reads are removed if read 1 had more than four non-T bases in the last ten bases (to remove all non-poly(T)-captured contaminated reads) Paired-end reads are removed if read 1 had one or more bases with a poor quality score (The right mate of each read pair is trimmed to remove any portion of the SMART adaptor sequence or any large stretches of poly(A) tails (6 consecutive bp or larger)The trimmed reads are aligned to the human genome (GENCODE GRCH38) with STAR (e.g. v2.5) with default parameter settingsReads that mapped to intronic or exonic regions as per the GENCODE gene annotation are included in gene countsBarcode synthesis errors are corrected by inserting N at the last base of the cell barcode for reads in which the first 11 bases of the cell barcode are identical and the last T base of the UMI is the sameUMI counts for each gene of each nucleus are assigned by collapsing UMI reads that had only 1 edit distance to create a digital expression matrix (genes as rows, cells or nuclei as columns)