Single cell 3 chip

The scRNA-seq libraries were generated using the Chromium Single Cell 3′Reagent Kit (10× Genomics). Briefly, a single-cell suspension (500 cells/μL in PBS) was mixed with real-time quantitative (RT-PCR) master mix and loaded together with Single Cell 3′Gel Beads and Partitioning Oil into a Single Cell 3′Chip (10× Genomics) according to the manufacturer’s instructions. RNA transcripts from single cells were uniquely barcoded and reverse transcribed within droplets. cDNA molecules were preamplified and pooled followed by library construction. All libraries were quantified on a 2100 Bioanalyzer (Agilent) with RT-PCR and then subjected to 150-bp paired-end sequencing on an Illumina Xten platform (sequenced by Novogene).

Single-cell suspensions were loaded onto 10x Genomics Single Cell 3′ Chips along with the reverse transcription (RT) mastermix per the manufacturer's protocol for the Chromium Single Cell 3′ Library (10x Genomics; PN-120233) to generate single-cell gel beads in emulsion (GEMs). Reverse transcription was performed using a C1000 Touch Thermal Cycler with a Deep Well Reaction Module (Bio-Rad) as follows: for 2 h at 55°C; for 5 min at 85°C; hold 4°C. cDNA was recovered and purified with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific; catalog no. 37002D) and SPRIselect beads (Beckman Coulter; catalog no. B23318). Purified cDNA was amplified as follows: for 3 min at 98°C; 12× (for 15 sec at 98°C; for 20 sec at 67°C; for 60 sec at 72°C); for 60 sec at 72°C; hold 4°C. Amplified cDNA was purified using SPRIselect beads and sheared to ∼200 bp with a Covaris S2 instrument (Covaris) using the manufacturer's recommended parameters. Sequencing libraries were generated with unique sample indices (SI) for each sample. Libraries for samples 1–3 and 4–5 were multiplexed, respectively, and sequenced on an Illumina NextSeq 500 (NextSeq control software v2.0.2/Real Time Analysis v2.4.11) using a 150-cycle NextSeq 500/550 High Output Reagent Kit v2 (Illumina, FC-404-2002) in standalone mode as follows: 98 bp (Read 1), 14 bp (I7 Index), 8 bp (I5 Index), and 10 bp (Read 2).