TITLE: 3-level sci RNA-Seq with FACS
AUTHORS: David Fraser Read, Cole Trapnell

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is a variation on "3 level sci RNA-Seq", presented on protocols.io at (</span><a href="https://www.protocols.io/view/sci-rna-seq3-9yih7ue" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/sci-rna-seq3-9yih7ue</span></a><span>).</span></div><div class = "text-block">The variation presented here notably includes a FACS sorting step before the PCR step. This notably decreases background in the library prep. </div></div>

[STEPS]
?. [Buffer Preparation]
Nuclei Buffer: 10 mM Tris HCl, 10 mM NaCl, 3 mM MgCl2 in nuclease-free waterStore at 4C.
?. [Buffer Preparation]
Nuclei suspension buffer (NSB).Prepare at least 1.7 mL per sample, plus 3 mL per RT plate and 1 mL per ligation plate. For every 1 mL. 1 mL Nuclei Buffer (10 mM Tris HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2)10 uL Bovine Serum Albumin (BSA)10 uL SuperaseInChill on ice
?. [Buffer Preparation]
10% Triton X-100 StockCombine 1.0 mL of Triton X-100 with 9.0 mL nuclease-free water. Gently rock overnight to mix, store at 4C.
?. [Buffer Preparation]
Permeabilization Buffer:Make 500 uL per sample. For this, combine:12.5 uL of 10% stock of Triton X-100487.5 uL NSBPre-chill on ice.
?. [Permeabilization]
Thaw frozen aliquots of all samples using a 37C water bath. Watch carefully, and remove from water bath and place on ice as soon as the frozen buffer + nuclei mixture has thawed.
?. [Permeabilization]
For each sample, add 400 uL permeabilization buffer. Mix gently.
?. [Permeabilization]
Incubate 3 minutes on ice.
?. [Permeabilization]
Pellet the nuclei at 500g for 5min (4C) and remove the supernatant; Resuspend the nuclei in 1 mL NSB
?. [Permeabilization]
Pellet the nuclei at 500g for 5min (4C) and remove the supernatant.
?. [Permeabilization]
Resuspend the nuclei in 300ul NSB and count the nuclei concentration with a hemocytometer.
?. [Reverse Transcription]
For each well of 4 x 96 well plates, add 30,000 nuclei in 22ul nuclei buffer2ul 10mM dNTP2ul indexed oligo-dT primer (100uM).Note: Up to 80,000 nuclei can be loaded per well. However, typical UMIs-per-nucleus decrease above ~30,000 nuclei per well.
?. [Reverse Transcription]
Incubate the plates at 55 °C for 5 min. Immediately place the plates on ice.
?. [Reverse Transcription]
Prepare the reverse transcription reaction mix. For each well, combine8 uL of 5X Superscript IV First-Strand Buffer2 uL of 100mM DTT2 uL of SuperScript IV reverse transcriptase2 uL of RNaseOUT Recombinant Ribonuclease InhibitorMix well and distribute 14ul to each well.Note: Make a master mix with a few extra wells worth to space. For example, consider making 105 or 110 wells worth of master mix in order to readily add 14 uL to each well of a 96 well plate.
?. [Reverse Transcription]
Incubate the RT reaction for the following times/temperatures:4°C for 2 minutes10°C for 2 minutes20°C for 2 minutes30°C for 2 minutes40°C for 2 minutes50°C for 2 minutes53°C for 15 minutes55°C for 10 minutes
?. [Reverse Transcription]
After the reaction, add 60ul ice-cold NBB into each well with a multichannel pipette.
?. [Reverse Transcription]
Pool the nuclei from all wells.
?. [Reverse Transcription]
Pellet the nuclei (600 RCF, 10min, 4C), and remove the supernatant.
?. [Ligation]
Resuspend the nuclei pellet in 1 mL NSB, then distribute 10 uL to each well of a 96 well plate.
?. [Ligation]
Add 8ul indexed ligation oligos (100 uM) into each well with a multichannel pipette.
?. [Ligation]
Prepare ligation master mix. For each well, combine:2 uL Quick Ligase20 uL Quick Ligase buffer
?. [Ligation]
Distribute 22 uL of ligation master mix to each well. Mix by gently pipetting up/down 5 times.
?. [Ligation]
Ligate by incubating at 25C for 10 minutes.
?. [Ligation]
Add 60 uL of NBB to each well. Pool all wells.
?. [Ligation]
Add an additional 10 mL NBB. Spin down nuclei (600 RCF, 10 minutes, 4C) and discard supernatant.
?. [Ligation]
Resuspend nuclei in 1 mL ice cold Elution Buffer ("EB", Qiagen).
?. [Ligation]
Add 10 uL of 300 uM DAPI (diluted in water), gently mix.
?. [Ligation]
Filter through the cap of a FACS tube (35 uM).
?. [FACS Sorting]
Add 4 uL of EB to each well of 96 well plates for sorting.
?. [FACS Sorting]
Sort singleton nuclei based on DAPI profiles. Sort the number of nuclei to give an acceptable doublet rate.The number of nuclei sorted per well here depends on the acceptable doublet rate and the number of RT and ligation wells used in previous steps. If a single 96 well plate was used for both RT and ligation, for instance, sorting 750 nuclei per well at this step corresponds to a ~92% singleton rate. The singleton rate is found as:(1-[1/(RT_wells * ligation_wells)] )^(#sorted per PCR well)
?. [Second Strand Synthesis]
First, confirm final volume in wells of FACS-sorted nuclei. Volume should be ~12 uL (from 4 uL EB plus buffer from sorted nuclei). If this volume is markedly different, scale all input volumes for every step from here up until the elution step in the "bead cleanup" step. So if wells have 25 uL in them, in your plate, all volumes below should be doubled up until the elution step from Ampure beads. That volume should remain constant, as should all following volumes.
?. [Second Strand Synthesis]
Prepare second strand master mix. For each well, prepare:1.33 uL second strand buffer.67 uL second strand enzyme mixAdd 2 uL per well, mix thoroughly.
?. [Second Strand Synthesis]
Incubate for 16 °C for 3 hours.
?. [Second Strand Synthesis]
Briefly spin plates.Optional stopping point. Store at 4C overnight, or freeze at -20C for longer periods.
?. [Tagmentation]
Make 2x TD Buffer:1.2 mL tagmentation salt buffer ( 20 mM Tris pH 7.4, 10 mM MgCl2)300 uL dimethyl formamide
?. [Tagmentation]
Make tagmentation mix. For each well:12.5 uL 2x TD buffer.02 uL Tn5 (N7-only)
?. [Tagmentation]
Add 12.5 uL of second tagmentation mix to each well. Mix thoroughly.
?. [Tagmentation]
Incubate for 5 minutes at 55C.
?. [Tagmentation]
Immediately add 25 uL DNA binding buffer per well and incubate the mix at room temperature for 5 minutes.
?. [Ampure bead purification]
Add 50 uL of Ampure Beads (pre-equilibrated to room temperature for at least 30 minutes, and mixed well) to each well.
?. [Ampure bead purification]
Incubate at room temperature for 5 minutes. Transfer to magnet and incubate for 3 minutes more.
?. [Ampure bead purification]
Wash beads, using a multichannel pipette. Wash twice. Each time:Remove supernatant.Wash each well with ~150 uL of 80% ethanol (made fresh from 100% ethanol stock).
?. [Ampure bead purification]
After the second wash of ethanol is removed, remove the plate from the magnet and let air-dry briefly. Note: Typically the time that it takes to remove all ethanol from all wells means that the first wells to be emptied will have beads fairly well-dried by the time the last wells in a plate have been emptied. The most essential part is to make sure all supernatant has been thoroughly removed from all wells before removing the plate from the magnet.
?. [Ampure bead purification]
Resuspend beads with a multichannel pipette, adding 17 uL EB to each well (Qiagen). Mix well.
?. [Ampure bead purification]
Incubate for 3 minutes at room temperature, then transfer to the magnet. Incubate 5 minutes.
?. [Ampure bead purification]
Transfer 16 uL from each well into a new plate (making sure to keep all wells separate at all times, still).
?. [PCR]
For each well, add:2ul indexedP5 PCR primer (10uM)2ul P7 primer (10uM)20ul NEBnext master mix for PCR reaction
?. [PCR]
Carry out PCR using the following cycle settings:72°C for 5 min, 98°C for 30 sec, 17 cycles of (98°C for 10 sec, 66°C for 30 sec, 72°C for 30 sec) and a final 72°C for 5 min.
?. [Post-PCR Cleanup]
Pool all PCR wells. For 1 plate of PCR product (40 uL * 96 wells) add 8 mL of DNA binding buffer, and mix well.
?. [Post-PCR Cleanup]
Clean DNA using a Qiagen "Clean and Concentrate" kit. Use 4 tubes per 1 PCR plate of product being processed to speed the process.
?. [Post-PCR Cleanup]
Load ~600 uL of DNA binding buffer/PCR product mix at a time. Spin 12,000 RCF for 30s, and discard flow-through. Repeat until all volume has been spun through filter tubes.
?. [Post-PCR Cleanup]
Wash 2x by adding 200 uL DNA wash buffer, and spinning 12,000 RCF for 30s.
?. [Post-PCR Cleanup]
Transfer filter tubes to sit in clean Eppendorf tubes. Add 52 uL EB carefully but directly onto the filter of each tube.
?. [Post-PCR Cleanup]
Incubate 1 minute at room temperature, then spin 12,000 RCF for 1 minute.
?. [Post-PCR Cleanup]
Combine all flow-through into a single tube (should be ~200 uL, from ~50 uL from each of 4 filter tubes in the previous step).
?. [Post-PCR Cleanup]
Carry out an Ampure bead cleanup using .7x volumes of bead volume (check volume eluted from column. For example, if 200 uL is eluted in the previous step add 140 uL of well-mixed, room temperature beads).
?. [Post-PCR Cleanup]
Incubate 5 minutes, move to magnet, incubate 3 minutes.
?. [Post-PCR Cleanup]
Remove supernatant and wash 2x with 80% fresh made ethanol. 1 mL each wash.
?. [Post-PCR Cleanup]
After removing second ethanol wash, air dry beads for 5 minutes.
?. [Post-PCR Cleanup]
Resuspend beads by adding 52 uL EB. Incubate for 5 minutes, transfer to magnet and incubate 3 minutes more. Remove 50 uL of solution, transfer and store as library.
?. [Quantify and Sequence]
Libraries were quantified by Qubit and fragment sizes/molar concentration was found using a D1000 kit on the Agilent ScreenTape.
?. [Quantify and Sequence]
Libraries were diluted and sequenced on an Illumina Nextseq 2000, 100 bp kitRead settings:Read 1: 34 basesRead 2 66 basesIndex 1: 10 basesIndex 2: 10 bases
?. [Buffer Preparation]
Nuclei buffer with BSA (NBB)Make at least: 6 mL per RT plate and 17 mL per ligation plate.For every 1 mL. 1 mL Nuclei Buffer (10 mM Tris HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2)10 uL Bovine Serum Albumin (BSA)Chill on ice
?. Note: All amounts listed here assume using 1 RT plate (96 wells) and 1 ligation plate (96 wells). Buffer volumes to prepare will vary for larger/smaller experiments.