GSM7882506: STB-CT30 snRNAseq; Homo sapiens; RNA-Seq
For STB-BL and STB-CT30 cell lines snRNA-seq sample extraction, cells were harvested and dissociated with TrypLE for 8 min at 37℃ and washed with phosphate buffer solution. Ice cold Nuclei EZ lysis buffer was added to resuspend the cells and grinded with a dounce tissue grinder. The lysates were bathed in ice and lysed twice. After washing with PBS, the nuclei were filtered through a 40 μm sieve to proceed RNA-seq. For hTSC-BL and hTSC-CT30 cell lines scRNA-seq sample extraction, cells were dissociated with TrypLE for 8 min at 37℃ and washed with phosphate buffer solution. For Single-nucleus RNA sequencing, 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% Bovine serum albumin (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, Beijing). Next, single-nucleus RNA-Seq library was constructed using Single Cell 3' Library and Gel Bead Kit V3.1 according to the manufacturer's introduction. The libraries were finally sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per nuclei with a pair-end 150bp (PE150) reading strategy (performed by CapitalBio Technology, Beijing). For Single-cell RNA Sequencing, the construction of transcriptome libraries was performed following the Chromium Single Cell 3' v2 Protocol. Briefly, the cDNA was purified after GEM generation, barcode addition, GEM-RT reaction and bead cleanup. The GEX libraries were generated following the 10 x Genomics Chromium Single Cell 3' Reagent Kits (v3) Protocol. Qubit 3.0 Fluorometer and Agilent 2100 High Sensitivity DNA Assay Kit were used to determine the library concentration and the distribution of library product fragments, respectively. Libraries were sequenced on the Illumina Novaseq 6000 platform with paired-end sequencing.