Single-Cell RNA-Seq Library Preparation

Libraries for scRNA-seq were prepared by using the Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (PN-1000075, 10 × Genomics, Pleasanton, CA, USA); Chromium Single Cell B Chip Kit (PN-1000073, 10 × Genomics); and Chromium i7 Multiplex Kit (PN-120262, 10 × Genomics). Cells were resuspended in PBS containing 0.04% BSA and diluted to ~ 2 × 10⁵ to ~ 1 × 10⁶ cells/mL. Cells were mixed with a reverse-transcription master mix and loaded onto B chip channels to capture ~ 800 to ~ 5000 single-cell transcriptomes. Gel bead-in emulsions (GEMs) were generated by using Chromium Controller (10 × Genomics). Reverse transcription was conducted by using a C1000 Touch thermal cycler (Bio-Rad, Hercules, CA, USA). DNA was purified, and libraries were constructed according to the manufacturer’s instruction. The qualities of amplified cDNAs and of the constructed libraries were assessed by using Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries were sequenced with a 2 × 100-bp paired-end protocol on a Novaseq-6000 platform (Illumina, San Diego, CA, USA) to generate at least 40,000 read pairs per cell. The sequencing depth recommended by the manufacturer for the 3′ Gene Expression library is a minimum of 20,000 read pairs per cell, and values in the range of 20,000 to 50,000 read pairs per cell are commonly used in the field [37 (link), 38 (link)].