Single cell b chip kit

Cell suspensions were loaded on a chromium single-cell controller (10× Genomics, San Francisco, CA, USA) to generate single-cell gel beads in emulsion (GEMs). A Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (1000121; 10× Genomics, Pleasanton, CA, USA) and Single-Cell B Chip Kit (1000127; 10× Genomics) were used according to the manufacturer’s protocol. As previously described, single-cell droplet collection, cDNA amplification, and sequencing library preparation were performed [46 (link),47 (link)]. The cDNA libraries were sequenced on an Illumina Novaseq6000 sequencer with a sequencing depth of at least 30,000 reads per cell and a pair end of 150 bp (PE150; Capitalbio Technology Corporation, Beijing, China).

Single Cell B Chip Kit (10x Genomics, 1000074) and the nucleus suspension (600 nuclei per microliter determined by CountStar) were loaded onto the Chromium single cell controller (10x Genomics) to generate single-nucleus gel beads in the emulsion (GEMs) according to the manufacturer’s protocol. In brief, single nuclei were suspended in PBS containing 0.04% BSA. About 16,000 nuclei were added to each channel, with 8,000 target nuclei estimated to be recovered. Captured nuclei were lysed, and the released RNA was barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on an S1000TM Touch Thermal Cycler (Bio-Rad) at 53 °C for 45 min, followed by 85 °C for 5 min and held at 4 °C. The cDNA was generated and then amplified, and quality was assessed using an Agilent 4200 (performed by CapitalBio Technology). Next, snRNA-seq library was constructed using Single Cell 3′ Library and Gel Bead Kit V3.1 according to the manufacturer’s instructions. The libraries were finally sequenced using an Illumina NovaSeq 6000 sequencer with a sequencing depth of at least 100,000 reads per nuclei with a pair-end 150 bp (PE150) reading strategy (performed by CapitalBio Technology).

Cell capture and cDNA synthesis were performed using a Chromium Single-Cell 3’ Gene Expression library and Gel Bead Kit V3.1 (10x Genomics, 1000075). Cell profiling was performed using a Single-Cell B Chip Kit (10x Genomics, 1000074). Cell suspension containing 300–600 living cells/μL (determined using Count Star) was loaded onto the Chromium single-cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer’s protocol. In brief, single cells were suspended in PBS containing 0.04% bovine serum albumin.
Around 8,700 cells were added to each channel with a targeted cell recovery estimate of 8,000 cells. Captured cells were lysed and the released RNA was barcoded through reverse transcription in individual GEMs (Gel bead-In-EMulsions). GEMs were reverse transcribed in a C1000 Touch Thermal Cycler (Bio Rad) programmed at 53°C for 45 min, 85°C for 5 min, and held at 4°C. After reverse transcription, single-cell droplets were broken, and single-stranded cDNA was isolated and cleaned with Cleanup Mix containing DynaBeads (Thermo Fisher Scientific). cDNA was generated and amplified, and the quality was assessed using the Agilent 4200.