TITLE: Single cell RNA sequencing library preparation (2-level sci-RNA-seq)
AUTHORS: Sanjay Srivatsan, Junyue Cao

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Sci-RNA-seq is a protocol developed by Junyue Cao in the Shendure Lab at the University of Washington and adapted by others in the Shendure and Trapnell labs. The protocol  It is among a family of protocols that leverages split-pool barcoding for single cell sequencing. </div></div>

[STEPS]
?. [Preparation]
Reagents to prepare prior to beginning protocol:1.  Prepare 5mL of NSBNSB - Nuclei Buffer + Superase In + BSA-------------------------------10mM Tris/HCl pH 7.410mM NaCl3mM MgCl21% (v./v) Superase-Inhibitor 1% (vol./vol.) BSASolvent: Nuclease Free Water2. Prepare 5mL of NBBNBB - Nuclei Buffer + BSA-------------------------------10mM Tris/HCl pH 7.410mM NaCl3mM MgCl21% (vol./vol.) BSASolvent: Nuclease Free Water
?. [Preparation]
Reagents purchased prior to beginning protocol:3.Each Superscript IV tube is sufficient for 192 barcoded RT reactions (2 plates). To increase the cell barcode space, more unique barcoded RT reactions must be performed.Each kit includes 5x SSIV buffer master mix and 0.1mM DTT necessary for performing RT reaction.4. Each tube is sufficient for ~495 barcoded RT reactions (4.5 plates).5. Alternatively, Tn5 can be produced and loaded with sequencing adapters in house. 6. 7. 8. 9. 10.
?. [Washing Nuclei]
Thaw the flash frozen nuclei on ice.---------------------------------------------------------------------------------------------------------------------To speed this step up hold tube in your hand and move the liquid by flicking the tube.
?. [Performing Reverse Transcription]
Set aside 220uL of nuclei per RT plate used.
?. [Second Strand Synthesis]
Perform second strand synthesis at 16C (with an unheated lid) for 150 minutes on a thermocycler.------------------------Plates can be stored at -20C after second strand synthesis for up to one month.
?. [Second Strand Synthesis]
Thaw plates on ice, and prepare a mixture of 0.5uL of Second Strand Synthesis Buffer and 0.25uL of Second Strand Synthesis enzyme mix for every well. ------------------------Making 10% overage can prevent frustration when adding mix to the final wells of a plate.
?. [Tagementation]
Prepare tagementation mix:5.75uL of 2x TD buffer (can be made at home)0.01uL of Tn5 n7 (made in house) or dual loaded Tn5 from IlluminaPrepare 10% overage (enough for 110 wells) to account for loss.
?. [Tagementation]
Start a 5 minute thermocycler program to tagment cDNA1. 5 minutes at 55C2. Forever at 4C-- Heated Lid
?. [SPRI cleanup and PCR]
After tagmentation, remove 96-well plate from the thermocycler and add 12uL of Zymo binding buffer to every well  . Mix well and allow the plate to sit at room temperature for 5 minutes
?. [Purifying Library and Sequencing]
After PCR,  pool samples and purify using a 1x AMPure bead cleanup
?. [Washing Nuclei]
Spin nuclei down at 800g for 6 minutes in a chilled (4C) swinging bucket centrifuge.
?. [Washing Nuclei]
Remove supernatant and resuspend nuclei in 100uL of NSB buffer. After the cells have been resuspended, add 400uL of permeabilization solution and pipette up and down gently to mix. Allow the cells to sit on ice for 3 minutes.
?. [Washing Nuclei]
Prepare permeabilization solution:390uL of NSB + 10uL of 10% Triton-X---------------------------------------------------------------------------------------------------------------------Keep this solution (and all others) cold on ice.
?. [Washing Nuclei]
Spin nuclei down at 800g for 6 minutes in a chilled (4C) swinging bucket centrifuge.
?. [Washing Nuclei]
Remove supernatant and resuspend nuclei in 400uL of NSB buffer. Use a sonicator to dislodge any aggregated nuclei or tissue. We use the bioruptor (https://www.diagenode.com/en/p/bioruptor-plus-sonication-device) on low for 12 seconds.
?. [Washing Nuclei]
Remove 10uL of nuclei and count cells using a hemocytometer. Using trypan blue is fine if the suspension is without significant cellular debris. Otherwise using DAPI as a nuclear stain in conjunction with a fluorescent microscope is very helpful.
?. [Washing Nuclei]
After counting adjust the nuclei concentration to 2500 nuclei/uL (or 2.5million nuclei per mL). ---------------------------------------------------------------------------------------------------------------------We have observed that overloading nuclei at this phase has led to detrimental results.
?. [Performing Reverse Transcription]
Add  0.25uL of dNTPs ()  for every 2uL of nuclei suspension.
?. [Performing Reverse Transcription]
Dispense 2.25uL of dNTP-nuclei suspension to each well of a 96 well low-bind PCR plate. ---------------------------------------------------------------------------------------------------------------------Using a multichannel and 8-strip PCR tube makes this process much easier. Make sure suspension stays cold while aliquoting.
?. [Performing Reverse Transcription]
Add 1.0uL of 10uM barcoded RT primer to each well.---------------------------------------------------------------------------------------------------------------------Spin down barcoded RT-Oligo plate prior to use to bring down any condensate that may have formed.Each RT primer contains a unique barcode sequence. Be careful when opening sealed plate. Use a different tip to add each RT primer and make sure no cross contamination occurs in stock oligo plate or in sample.
?. [Performing Reverse Transcription]
Perform an annealing step on a thermocycler:1. 5 minutes at 55°C (heated lid).2. Forever at 4°C After this cycle is finished, move back onto ice immediately.
?. [Performing Reverse Transcription]
Prepare RT Master Mix for every well:Per Well:1uL 5x SSIV Master Mix0.25uL of 0.1mM DTT0.25 uL RNseOUT0.25 uL of SSIV RTPer Plate (includes 10% overage):110uL 5x SSIV Master Mix27.5 uL of 0.1mM DTT27.5 uL RNseOUT27.5 uL of SSIV RTMix well after combining. Solutions are very viscous.
?. [Performing Reverse Transcription]
After annealing, dispense 1.75uL of RT Master Mix (made in step 16), into each well and mix. Seal with a plate sealer. ---------------------------------------------------------------------------------------------------------------------Again, it is important to keep every well completely independent. Reagent can be added robotically and mixed with a pulse on a benchtop vortexer.
?. [Preparation]
Specific consumables needed:1. 2. Multi-channel pipettes (12 channel or 8 channel; 1uL range 20uL range and 200uL range all very useful)3. 4. 5. Thermocyler with 96 well heating block6. Temperature controlled swinging bucket bench top centrifuge
?. [Performing Reverse Transcription]
Start the reverse transcription reaction on the thermocycler:1. 2 minutes at 4°C, 2. 2 minutes at 10°C 3. 2 minutes at 20°C4. 2 minutes at 30°C5. 2 minutes at 40°C6. 2 minutes at 50°C7. 15 minutes at 55°C8. Forever at 4°C -- Heated Lid
?. [Performing Reverse Transcription]
After reverse transcription is finished, place plate on ice and add 15uL of NBB (Nuclei Buffer + BSA) to every well. Pool all the wells into a trough. Place pooled suspension into a FACs tube and stain nuclei by adding 5uL of 100uM DAPI. ---------------------------------------------------------------------------------------------------------------------Note: It takes 5-10minutes for DAPI to fully intercalate.While reverse transcription is happening thaw reagents for second strand synthesis.
?. [FACs Sorting (Option 1)]
Calibrate the flow sorter to dispense into a 96 well plate. We normally sort on a BD Aria 3 and we use an 80um nozzle, and a 50um filter on the inlet. We also have the sample chilled. Using the same type of PCR plate that you will be sorting into with a seal on, calibrate the machine so that you can sort into the center of each well.
?. [FACs Sorting -- op]
Spin down the pool of nuclei at 650g for 6 minutes in a cooled swinging bucket centrifuge.
?. [FACs Sorting -- op]
Carefully aspirate supernatant and resuspend nuclei in 100uL
?. [PCR]
To each well add 2uL of indexed P7 and 2uL of indexed P5 PCR primers.The combination of P5 and P7 indices used for each well must be unique. Crucially this forms the second level of molecular indexing
?. [FACs Sorting (Option 1)]
Set up low-bind 96 well plates prior to FACs sorting. Each well in the prepared plates should contain 5uL of Elution Buffer. -----For a subset of the plates, if you are planning on performing second strand synthesis, we recommend mixing 0.5uL of Second Strand Synthesis Buffer and 0.25uL of Second Strand Synthesis enzyme mix with EB prior to addition to a 96 well plate
?. The likelihood of two cells receiving the same set of barcodes is defined by the birthday problem; the probability that two people in a group of a certain size will have the same birthday. Analogous to the birthday problem, the more cells that are sorted into a PCR well, the higher the likelihood that two or more cells came from the same indexed reverse transcriptase well.This means that increasing the number of RT wells used, will decrease the likelihood of a barcode collision and control the doublet rate. Prior to beginning an experiment, choose a target number of cells you would like to recover upo n sequencing. The attached image from Cusanovich et. al. 2015 details how to calculate the likelihood of a collision.A good rule of thumb for 2-level sci-RNA-seq is that every 96 RT-barcodes allows you to sort 25 cells/well at a 10% doublet rate. Therefore after library preparation we would expect 25*96 cells (2400) cells per sequenced plate.In practice this number turns out to be between 65%-75% of this value, due to the loss of cells that form poor libraries.
?. [FACs Sorting (Option 1)]
After calibrating the sorter, find the DAPI stained nuclei. We adjust the voltage to set the main DAPI peak (marking cells in G1/S) at 50. Then, while viewing the nuclei in DAPI - Area versus DAPI - height, we draw a polygon to capture singlets, or those cells that are on-diagonal (nuclei for which the DAPI area is equal to the DAPI height)
?. [FACs Sorting (Option 1)]
Sort the number of cells/well determined in step#4
?. [FACs Sorting (Option 1)]
After sorting, spin plates down briefly to bring down any cells that may be stuck to the walls of the 96-well plate. Begin second strand synthesis on any plates that contain second strand synthesis reagent. Otherwise plates can be stored at 4C for 2-3 days or at -80C for  2 weeks to a month.
?. [Dilution (Option 2)]
Diluting nuclei is faster way of seeding nuclei, but dilution can lead to more background RNA and a higher number of physical doublets. The latter is dependent on how completely cells were disassociated
?. [Dilution (Option 2)]
After pooling nuclei from RT PCR, spin nuclei down in a swinging bucket centrifuge at 650g for 5minutes with the temperature set to 4C.
?. [Dilution (Option 2)]
Carefully remove the supernatant, being careful not to disturb the pellet.
?. [Dilution (Option 2)]
Resuspend nuclei in 250uL of NSB and count the concentration of nuclei per uL using a hemocytometer and trypan blue.  If the nuclei are small or hard to localize, try staining with DAPI prior to counting. Finally, if the concentration of nuclei is too high repeat procedure with a 1:10 or 1:100 dilution
?. [Dilution (Option 2)]
With the final count in hand resuspend nuclei to the desired final concentration, using NSB as the diluent. Each well will receive 5uL of the nucleus suspension so the concentration of the diluted solution should be one-fifth the number of nuclei desired per well.For example if 25 nuclei/well is the target, the desired concentration is 5nuclei/uL
?. [Dilution (Option 2)]
For a subset of the plates, if you are planning on performing second strand synthesis right away, we recommend mixing 0.5uL of Second Strand Synthesis Buffer and 0.25uL of Second Strand Synthesis enzyme mix with the diluted nucleus suspension prior to addition to a 96 well plate If you choose this route load 5.75uL of Second Strand Synthesis + Nucleus suspension into each well of a 96 well plate and proceed to step #33
?. [Dilution (Option 2)]
Aliquot out 5uL of diluted nucleus suspension in 96 well low-bind plates.Plates can be stored at 4C for 2-3 days or at -80C for  2 weeks to a month.
?. [Dilution (Option 2)]
If there are a lot of clumps or aggregates present, pass the nucleus suspension through a mesh strainer to remove large clumps. We prefer to use BD FACs tubes that contain a 40um mesh built into the cap. Passing the nuclei through this strainer should separate out large clumps.
?. [Second Strand Synthesis]
Add 0.75uL of the second strand synthesis enzyme + buffer mix to each well. Seal with a plate seal  and mix by gently vortexing. Spin nuclei down prior starting the next step.
?. [Tagementation]
Keeping the tagmentation mix on ice, remove the nuclei from the thermocycler and add 5.75uL of the tagmentation mix to every well.Make sure to mix throughly.
?. [SPRI cleanup and PCR]
Prior to using SPRI beads, make sure they have been warmed to room temperature
?. [SPRI cleanup and PCR]
Add 36uL of SPRI beads to each well, mixing to make sure that the solutions are completely mixed.Allow this to sit for 5 minutes before placing on a magnetic stand.
?. [SPRI cleanup and PCR]
Remove unbound material from each well by removing the supernatant from every well.
?. [SPRI cleanup and PCR]
Add 100uL of 75% ethanol to wash beads. Wait 30 seconds, and then remove.Perform this wash twice.
?. [SPRI cleanup and PCR]
After removal of the second ethanol wash allow wells to dry for 3-4 minutes.Be careful at this stage, wet beads lead to the carryover of ethanol a PCR inhibitor when present at sufficiently high quantities. On the other hand overly dried beads crack and do not reconstitute as well.
?. [SPRI cleanup and PCR]
After 3-4minutes of drying add 17uL of pre-PCR molecular grade water to each well. Resuspend each well getting all the beads back into solution.
?. [SPRI cleanup and PCR]
Place 96-well plate on a magnet and transfer 16uL of eluted, tagmented cDNA into a new 96 well plate.
?. [SPRI cleanup and PCR]
All wells must be kept independent from one another. Cross contamination between wells can lead to cross contamination of RNA molecules between different cells. Use a separate pipette for every well. ------------------------To reduce tip waste we generally save the tip used for each well by placing the tip back into the tip box. This allows for the tip to be used during mixing the beads, removal of the unbound DNA, ethanol washes and transfer of the eluted DNA.
?. [PCR]
Add 20uL of 2x NEB Next MasterMix to everywell and mix
?. [PCR]
Each well should contain:2μL of 10 μM P5 primer (5′-AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′), 2 μL of 10 μM P7 primer (5′-CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG-3′),20 μL NEBNext High-Fidelity 2X PCR Master Mix (NEB)
?. [PCR]
Seal the plate and briefly centrifuge
?. [PCR]
PCR amplification was carried out using the following program: 72°C for 5 min,98°C for 30 sec98°C for 10 sec66°C for 30 sec72°C for 30 secReturn to step 3 for 18-22 cycles72°C for 5 min.
?. [PCR]
PCR product can be stored at -20C or 4C for a reasonable amount of time.
?. [Purifying Library and Sequencing]
Run samples on a TapeStaion, BioA or PAGE gel to estimate the average library size
?. [Purifying Library and Sequencing]
Use a Qubit to calculate the concentration of DNA
?. [Purifying Library and Sequencing]
Using the Qubit concentration and the size from the TapeStation, calculate the molarity
?. [Purifying Library and Sequencing]
Samples can be sequenced on any sequencing platform with the following read lengths:R1: 18I1: 10I2 :10R2 : 52For NextSeq550 and NextSeq2000 runs we recommend a loading concentration of 2pM and 650pM respectively.