Chromium single cell b chip kit

Using single cell 3′ GEM, Library & Gel Bead Kit V3.1 (10× Genomics, 1000075) and Chromium Single Cell B Chip Kit (10× Genomics, 1000074), the prepared cell suspension (300–600 living cells per μL determined by Count Star) was loaded onto the Chromium single cell controller (10× Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. Then single cells were suspended in PBS containing 0.04% BSA. The target cell will be recovered to about 8000 cells by estimation. Captured cells were lysed to release their RNA, which were then barcoded through reverse transcription in individual GEMs. Then reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53°C for 45 min, followed by 85°C for 5 min. The cDNA was kept at 4°C, then was amplified and the quality was assessed using an Agilent 4200 (performed by CapitalBio Technology, Beijing).

Each 10,000 nuclei were used for the snRNA or snATAC library construction.
For snRNA-seq, the single cell 3′ GEM, Library & Gel Bead Kit V3.1 (10× Genomics, 1000075) and Chromium Single Cell B Chip Kit (10× Genomics, 1000074) were used. To generate single-nuclei gel beads in emulsion, the nuclei suspension was loaded onto the Chromium single cell controller (10× Genomics). Then suspended the single nuclei in PBS (containing 0.04% BSA). Captured cells were lysed to release their RNA and barcoded through reverse transcription in individual GEMs. The reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio-Rad) at 53 °C for 45 min, followed by 85 °C for 5 min. The cDNA was kept at 4 °C and then amplified for sequencing.
For snATAC-seq, incubating the nuclei with Tn5 transposase. Then the nuclei suspension was loaded into the Chromium microfluidic chip E with 10x Genomics reagents and barcoded with a 10x Genomics Chromium Controller (10x Genomics, Pleasanton, CA). DNA fragments were subsequently amplified, and the sequencing libraries were constructed with reagents from a Chromium Single Cell ATAC reagent kit (10x Genomics; PN-1000110, PN-1000156, PN-1000084) according to the manufacturer’s instructions. After preliminary quantification and quality inspection, libraries were then pooled and loaded on an Illumina NovaSeq with 2 × 50 paired-end kits.

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)].