Single cell 5 library and gel bead kit

Using the Single Cell 5’ Library and Gel Bead Kit (10X Genomics, 120237) and Chromium Single Cell A Chip Kit (10X Genomics, 120236), the cell suspension was loaded onto the Chromium single-cell controller (10X Genomics) to generate single-cell gel beads in the emulsion (GEMs) according to the manufacturer’s protocol. Briefly, single cells were suspended in PBS containing 0.04% bovine serum albumin. Approximately 10,000 cells were added to each channel, and about 6000 cells were recovered. The captured cells were lysed, and the released RNA was barcoded via reverse transcription in individual GEMs. Reverse transcription was performed at 53°C for 45 min, followed by 85°C for 5 min, and then the temperature was held at 4°C in a C1000 Touch Thermal Cycler (Bio Rad). After reverse transcription, single-cell droplets were broken and the single-strand cDNA was isolated and cleaned with Cleanup Mix containing DynaBeads (Thermo Fisher Scientific). cDNA was generated and amplified, and quality was assessed using the Agilent 4200. Single-cell RNA-seq libraries were prepared using Single Cell 5’ Library Gel Bead Kit V2 following the manufacture’s introduction. Next generation sequencing was performed on an Illumina Novaseq6000 with a sequencing depth of at least 100,000 reads per cell and pair end 150 bp (performed by CapitalBio Technology, Beijing).