GSM4569036: 10938438_5; Mus musculus; RNA-Seq
Single cells were FACS sorted into ice-cold 96-well PCR plates (Thermofisher) containing 2 µl lysis mix per well. The lysis mix contained 0.5 µl 0.4% (v/v) Triton X-100 (Sigma-Aldrich), 0.05 µl 40 U/µl RnaseOUT (Thermofisher), 0.08 µl 25 mM dNTP mix (Thermofisher), 0.5 µl 10 µM (dT)30_Smarter primer (Table S3), 0.05 µl 0.5 pg/µl External RNA Controls Consortium (ERCC) spike-ins mix (Thermofisher), and 0.82 µl PCR-grade H20 (Qiagen). Immediately after cell sorting, each plate was covered with adhesive film (Thermofisher), briefly spun down in a benchtop plate centrifuge, and frozen on dry ice. Plates containing single cells in lysis mix were stored at -80°C and shipped on dry ice (only T cells) until further processing. The plate containing single cells in lysis mix was thawed on ice, briefly spun down in a benchtop plate centrifuge, and incubated in a thermal cycler for 3 minutes at 72°C (lid temperature 72°C). Immediately after, the plate was placed back on ice and 3 µl RT mastermix was added to each well. The RT mastermix contained 0.25 µl 200 U/µl SuperScript II (Thermofisher), 0.25 µl 40 U/µl RnaseOUT (Thermofisher), and 2.5 µl 2x RT mastermix. The 2x RT mastermix contained 1 µl 5x SuperScript II buffer (Thermofisher), 0.25 µl 100 mM DTT (Thermofisher), 1 µl 5 M betaine (Sigma-Aldrich), 0.03 µl 1 M MgCl2 (Sigma-Aldrich), 0.125 µl 100 µM well-specific template switching oligonucleotide TSO_BCx_UMI5_TATA (Table S3 and Table S4), and 0.095 µl PCR-grade H2O (Qiagen). Reverse transcription was performed in a thermal cycler (lid temperature 70°C) by 90 min at 42°C, followed by 10 cycles of 2 min at 50°C and 2 min at 42°C, then 15 min at 70°C. Plates with single-cell cDNA were stored at -20°C until further processing. For cDNA amplification, 7.5 µl LD-PCR mastermix were added to each well. The LD-PCR mastermix contained 6.25 µl 2x KAPA HiFi HotStart ReadyMix (Roche Diagnostics), 0.125 µl 20:µM PCR_Satija forward primer (Table S3), 0.125 µl 20 µM SmarterR reverse primer8 (Table S3), and 1 µl PCR-grade H2O (Qiagen). The amplification was performed in a thermal cycler (lid temperature 98°C) by 3 min at 98°C, followed by 22 cycles of 15 sec at 98°C, 20 sec at 67°C, 6 min at 72°C, then a final elongation for 5 min at 72°C. Plates with amplified single-cell cDNA were stored at -20°C until further processing. For library preparation, 5 µl amplified cDNA from each well of a 96-well plate were pooled and completed to 500 µl with PCR-grade H2O (Qiagen). Two rounds of 0.6X solid-phase reversible immobilization beads (AmpureXP, Beckman, or CleanNGS, Proteigene) cleaning were used to purify 100 µl pooled cDNA with final elution in 15 µl PCR-grade H2O (Qiagen). After quantification with Qubit dsDNA HS assay (Thermofisher), 800 pg purified cDNA pool were processed with the Nextera XT DNA sample Preparation kit (Illumina), according to the manufacturer's instructions with modifications to enrich 5'-ends of tagmented cDNA during library PCR. After tagmentation and neutralization, 25 µl tagmented cDNA was amplified with 15 µl Nextera PCR Mastermix, 5 µl Nextera i5 primer (S5xx, Illumina), and 5 µl of a custom i7 primer mix (0.5 µM i7_BCx + 10 µM i7_primer, Table S3). The amplification was performed in a thermal cycler (lid temperature 72°C) by 3 min at 72°C, 30 sec at 95°C, followed by 12 cycles of 10 sec at 95°C, 30 sec at 55°C, 30 sec at 72°C, then a final elongation for 5 min at 72°C. The resulting library was purified with 0.8X solid-phase reversible immobilization beads (AmpureXP, Beckman, or CleanNGS, Proteigene). Libraries generated from multiple 96-well plates of single cells and carrying distinct i7 barcodes were pooled for sequencing on an Illumina NextSeq550 platform, with High Output 75 cycles flow cells,  targeting  5x105 reads per cell in paired-end single-index mode with the following primers (Table S3) and cycles: Read1 (Read1_SP, 67 cycles), Read i7 (i7_SP, 8 cycles), Read2 (Read2_SP, 16 cycles).