TITLE: sci-RNA-seq3
AUTHORS: Junyue Cao, Jay Shendure

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Single-cell combinatorial indexing (‘sci-’) is a methodological framework that employs split-pool barcoding to uniquely label the nucleic acid contents of large numbers of single cells or nuclei. Although the throughput of ‘sci-’ methods increases exponentially with the number of rounds of indexing, this potential has yet to be fully realized because of other factors such as the rate of cell loss and the limited reaction efficiency of some steps. To address this, we developed and extensively optimized 3-level sci-RNA-seq (sci-RNA-seq3), resulting in a workflow that can profile millions of cells per experiment. As previously multiple samples (e.g. replicates, timepoints, etc.) can be barcoded during the first round of indexing and concurrently processed.</div></div>

[STEPS]
?. [Buffer preparation]
500ml Nuclei buffer (Stored in 4C): 10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2 in nuclease-free water:  ABCD1ReagentStock concentrationFinal concentrationVolume (ml)2Tris-HCl (pH 7.5)1M10mM53NaCl5M10mM14MgCl21M3mM1.55Nuclease-free waterNANA492.56Final volume500Filter the buffer through 0.22uM filter and store the buffer in 4C for up to 1 year.
ABCD1ReagentStock concentrationFinal concentrationVolume (ml)2Tris-HCl (pH 7.5)1M10mM53NaCl5M10mM14MgCl21M3mM1.55Nuclease-free waterNANA492.56Final volume500
?. [Buffer preparation]
20ml 10% (volume) IGEPAL CA-630 in nuclease-free water (stored in 4C).Add 2ml IGEPAL CA-630 to 18ml nuclease-free water. Mix the solution by pipetting up and down for 20 times. The mix can be stored in 4C for up to 1 year.
?. [Buffer preparation]
20ml 10% (volume) Triton X-100 in nuclease-free water (stored in 4 degrees).Add 2ml Triton X-100 to 18ml nuclease-free water. Mix the solution by pipetting up and down for 20 times. The mix can be stored in 4C for up to 1 year.
?. [Buffer preparation]
Cell lysis buffer(CLB) (Made fresh each time, 5ml per sample, stored on ice): Nuclei buffer with 0.1% IGEPAL CA-630, 1% SUPERase In RNase Inhibitor (20 U/µL, Ambion) and 1% BSA (20 mg/ml, NEB). For each sample, combine 4.85ml nuclei buffer,  50ul 10% (volume) IGEPAL CA-630, 50ul SUPERase In RNase Inhibitor (20 U/µL, Ambion), and 50ul BSA (20 mg/ml, NEB).
?. [Buffer preparation]
Nuclei suspension buffer (NSB) (Made fresh each time, 3ml per sample, stored on ice): Nuclei buffer with 1% SUPERase In RNase Inhibitor (20 U/µL, Ambion) and 1% BSA (20 mg/ml, NEB). For each sample, combine 4.9ml nuclei buffer,  50ul SUPERase In RNase Inhibitor (20 U/µL, Ambion), and 50ul BSA (20 mg/ml, NEB).
?. [Nuclei extraction directly from tissues (~1.5 hours for 6 tissues)]
Cool the centrifuge to 4C.In a 6cm dish, add 1ml CLB to the frozen tissue (0.1g-0.5g, frozen in liquid nitrogen or -80C) for nuclei extraction. Cut the tissue into small pieces (
?. [Nuclei extraction directly from tissues (~1.5 hours for 6 tissues)]
Pipet the tissue up and down with 1ml pipet tip for 10 times (cut the top of the 1ml pipet tip for easy pipetting) and transfer the tissue into one 15ml tube with another 3ml CLB.
?. [Nuclei extraction directly from tissues (~1.5 hours for 6 tissues)]
Transfer the CLB with tissue to the top of a 40um cell strainer (placed on the top of 1 well in 6-well plate on ice) and homogenize the tissue with the rubber tip of a syringe plunger. (During the tissue dissociation, make sure the tissue on the strainer is merged in cold lysis buffer).
?. [Nuclei extraction directly from tissues (~1.5 hours for 6 tissues)]
Transfer the isolated and filtered nuclei into a new 15ml tube, pellet the nuclei (500g, 5min), and dump the supernatant.
?. [Nuclei extraction directly from tissues (~1.5 hours for 6 tissues)]
Re-suspend the nuclei in 1ml CLB by pipetting up and down 3 times.
?. [Nuclei fixation (~1.5 hours for 6 tissues)]
Pellet the nuclei (500g 5min) and dump the supernatant.
?. [Nuclei fixation (~1.5 hours for 6 tissues)]
Re-suspend the nuclei in 100ul NSB, add 10ml ice-cold 4% PFA, and invert the tubes for 5 times. Fix the nuclei on ice for 15min.
?. [Nuclei fixation (~1.5 hours for 6 tissues)]
Pellet the nuclei (500g, 3min) and dump supernatant.
?. [Nuclei fixation (~1.5 hours for 6 tissues)]
Re-suspend the nuclei with 1ml NSB and transfer the nuclei into a new tube. Pellet the nuclei (500g, 5min) and dump supernatant.
?. [Nuclei fixation (~1.5 hours for 6 tissues)]
Re-suspend the nuclei with 1ml NSB. Pellet the nuclei (500g, 5min) and remove supernatant as much as possible with pipetting.
?. [Nuclei fixation (~1.5 hours for 6 tissues)]
Re-suspend the nuclei with 500ul NSB. Split the nuclei into five tubes with 100ul each. Flash freeze the nuclei in liquid nitrogen and store the nuclei in liquid nitrogen or -80C freezer(stop point).
?. [Nuclei permeabilization before reverse transcription (1 hour for 6 samples)]
Preparation before the experiment: Cool the centrifuge to 4C.Prepare nuclei suspension buffer (NSB): for 25 samples, combine 40ml nuclei buffer, 400ul SUPERase In RNase Inhibitor (20 U/µL, Ambion), and 400ul BSA (20 mg/ml, NEB). Store the mixture on ice. Prepare nuclei buffer with 1% BSA (NBB): 100ml nuclei buffer and 1ml BSA (20 mg/ml, NEB). Store the buffer on ice.
?. [Nuclei permeabilization before reverse transcription (1 hour for 6 samples)]
Thaw paraformaldehyde (PFA) fixed nuclei (100ul flash-frozen in liquid nitrogen) in 37C water bath.
?. [Nuclei permeabilization before reverse transcription (1 hour for 6 samples)]
Pellet the nuclei at  500g for 5min (4C) and remove the supernatant.
?. [Nuclei permeabilization before reverse transcription (1 hour for 6 samples)]
Add 100ul NSB to resuspend the nuclei. For each sample, add mixture containing 390ul NSB  and 10ul 10% Triton X-100.
?. [Nuclei permeabilization before reverse transcription (1 hour for 6 samples)]
Incubate the mix on ice for 3min.
?. [Nuclei permeabilization before reverse transcription (1 hour for 6 samples)]
Pellet the nuclei at 500g for 5min (4C) and remove the supernatant; Resuspend the nuclei in 400ul NSB;
?. [Nuclei permeabilization before reverse transcription (1 hour for 6 samples)]
Perform sonication in the Bioruptor Sonication device (low power mode, 12s).
?. [Nuclei permeabilization before reverse transcription (1 hour for 6 samples)]
Filter the nuclei through theFlowmi cell strainer.
?. [Nuclei permeabilization before reverse transcription (1 hour for 6 samples)]
Pellet the nuclei at 500g for 5min (4C) and remove the supernatant;
?. [Nuclei permeabilization before reverse transcription (1 hour for 6 samples)]
Resuspend the nuclei in 100ul NSB and count the nuclei concentration with a hemocytometer.
?. [Reverse transcription (~ 2 hours for 6 samples)]
For each well of 4 x 96 well plates, add 80,000 nuclei in 22ul nuclei buffer (with wide bore tips); 2ul 10mM dNTP; and 2ul indexed oligo-dT primer (100uM);
?. [Reverse transcription (~ 2 hours for 6 samples)]
Incubate the plates at 55 °C for 5 min. Immediately place the plates on ice.
?. [Reverse transcription (~ 2 hours for 6 samples)]
Prepare the reverse transcription reaction mix by combining: 5X Superscript IV First-Strand Buffer: 3280ul 100mM DTT: 820ul SuperScript IV reverse transcriptase: 820ul RNaseOUT Recombinant Ribonuclease Inhibitor: 820ul Mix well and distribute 14ul to each well.
?. [Reverse transcription (~ 2 hours for 6 samples)]
Start the RT reaction with the following program: 4°C 2 minutes, 10°C 2 minutes, 20°C 2 minutes, 30°C 2 minutes, 40°C 2 minutes, 50°C 2 minutes and  55°C 15 minutes.
?. [Reverse transcription (~ 2 hours for 6 samples)]
After the reaction, add 60ul ice-cold NBB into each well with a multichannel pipette.
?. [Reverse transcription (~ 2 hours for 6 samples)]
Pellet the nuclei (500g, 10min), and remove the supernatant.
?. [Ligation (~ 2 hours)]
Resuspend the cells in 4.3ml NSB, and distribute 10ul into each well of 4 x 96well plates.
?. [Ligation (~ 2 hours)]
Add 8ul indexed ligation oligos (100 uM) into each well with a multichannel pipette.
?. [Ligation (~ 2 hours)]
Prepare ligation master mix by combining: Quick ligase buffer: 8.6ml; Quick ligase: 860ul; Mix well and distribute 22ul into each well with gently pipetting up and down five times (with wide bore tip).
?. [Ligation (~ 2 hours)]
Ligation at 25C for 10min.
?. [Ligation (~ 2 hours)]
add 60ul NBB into each well and pool the nuclei from all wells (with wide bore tips).
?. [Ligation (~ 2 hours)]
Add another 40ml NBB to the nuclei mix. Pellet the nuclei (600g, 10min) and dump the supernatant.
?. [Ligation (~ 2 hours)]
Resuspend the cells in 5ml NBB. Pellet the nuclei (600g, 10min) and dump the supernatant.
?. [Ligation (~ 2 hours)]
Resuspend cells in 4ml NBB. Filter the nuclei with the flowmi cell strainer (check the nuclei under the microscope and repeat the filtering step again if there are still a lot of doublets).
?. [Ligation (~ 2 hours)]
Count the nuclei concentration with a hemacytometer.
?. [Ligation (~ 2 hours)]
Distribute the diluted nuclei (in NBB) into several 96 well plates with 2500 to 4000 nuclei per well (5ul total volume).
?. [Ligation (~ 2 hours)]
Briefly spin the plates. The plates can be frozen in -80C for at least 1 month (stop point).
?. [Second strand synthesis (~ 3 hours)]
Thaw one plate (with nuclei distributed per well) in room temperature.
?. [Second strand synthesis (~ 3 hours)]
Prepare second strand synthesis mix: Combine 330ul elution buffer, 146.7ul second strand synthesis buffer, and 73.3ul second strand synthesis enzyme and mix well; Distribute 5ul reaction mix per well and briefly centrifuge the plate.
?. [Second strand synthesis (~ 3 hours)]
Perform second strand synthesis at 16C for 3 hours.
?. [Tagmentation (~10min)]
Prepare the tagmentation mix by combining 1.1ml tagmentation buffer, 2.75ul N7 adaptor loaded Tn5 and mix well. Briefly centrifuge the plate and add a 10ul reaction mix into each well.
?. [Tagmentation (~10min)]
perform tagmentation at 55C at 5min;
?. [Tagmentation (~10min)]
Add 20ul DNA binding buffer per well and incubate the reaction mix at room temperature for 5min.
?. [Ampure beads purification (~1 hour)]
Prepare USER mix: combine 880ul nuclease-free water, 110ul 10X USER buffer, 110ul USER enzyme (NEB) and mix well.
?. [Ampure beads purification (~1 hour)]
For each well, add 40ul Ampure XP beads and mix well. Incubate the mixture at room temperature for 5min and briefly centrifuge the plate.
?. [Ampure beads purification (~1 hour)]
Put the plate on the magnetic stand for 5min and remove the supernatant.
?. [Ampure beads purification (~1 hour)]
Wash each well with 100ul 80% ethanol twice.
?. [Ampure beads purification (~1 hour)]
After the second wash, carefully remove all supernatant from each well and dry the plate at room temperature for 3min.
?. [Ampure beads purification (~1 hour)]
For each well, add 10ul USER reaction mix and seal the plate. Vortex the plate to mix the beads with the USER reaction mix. Briefly centrifuge and incubate the plate at 37C for 15min.
?. [Ampure beads purification (~1 hour)]
Briefly centrifuge the plate. Add 7ul elution buffer to each well and mix well.
?. [Ampure beads purification (~1 hour)]
Briefly centrifuge the plate and leave the plate on the magnetic stand for 3min.
?. [Ampure beads purification (~1 hour)]
Transfer the supernatant (16ul) to a new 96 well plate (on ice).
?. [PCR reaction and library preparation (~2 hours)]
Briefly spin the plate.
?. [PCR reaction and library preparation (~2 hours)]
For each well, add 2ul P5 PCR primer (10uM), 2ul P7 primer (10uM) and 20ul NEBnext master mix for PCR reaction: 72°C for 5 min, 98°C for 30 sec, 12 to 15 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 (stop point).
?. [PCR reaction and library preparation (~2 hours)]
Pool all wells together, and take 1ml PCR product for a normal 0.7X Ampure XP beads purification. Elute the product in 100ul elution buffer.
?. [PCR reaction and library preparation (~2 hours)]
Perform a second round of 0.8x Ampure XP beads purification.
?. [PCR reaction and library preparation (~2 hours)]
Library concentrations were determined by Qubit (Invitrogen) and the libraries were visualized by electrophoresis on a 6% TBE-PAGE gel (example image shown below).
?. [PCR reaction and library preparation (~2 hours)]
Library was sequenced on Novaseq platform (Read1: 34bp, Read2: 100bp, index 1: 10bp, index2: 10bp).
?. [Reverse transcription (~ 2 hours for 6 samples)]
Pool the nuclei from all wells (with wide bore tip).