GSM8462147: AL12, scATAC-seq; Homo sapiens; ATAC-seq
We collected fresh tumor tissues which was mechanically and enzymatically dissociated using a gentleMACS dissociator (Miltenyi Biotec, Gladbach Bergisch, Germany, Cat#130-093-235) and the Human Tumor Dissociation Kit (Miltenyi Biotec, Cat#130-095-929) following the manufacturer's instructions. After incubation for 1 h at 37℃, the resuspended samples were filtered through a 70-µm MACS SmartStrainer (Miltenyi Biotec, Cat# 130-098-462) into RPMI-1640 medium (Corning, Inc. Corning, NY, USA) supplemented with 10% fetal bovine serum (Biowest, Riverside, MO, USA) and centrifuged at 300 ×g for 10 min. Single-cell RNA and TCR libraries were generated using the Chromium controller in accordance with the 10x Chromium Next GEM Single Cell 5-v2_CellSurfaceProtein_UserGuide (CG000330). Cell suspensions were initially diluted with nuclease-free water to a target concentration of 10,000 cells. These suspensions were then combined with the master mix, Single Cell 5′ Gel Beads, and Partitioning Oil, and loaded into a Next GEM Chip K. Within the droplets, RNA transcripts from individual cells were barcoded and reverse-transcribed. The resulting cDNA was pooled and subjected to PCR enrichment. Following amplification, the cDNA was size-selected to generate three distinct library types: 5ʹ Gene Expression, V(D)J Enriched, and Cell Surface Protein. For the Cell Surface Protein Library, the cDNA pool underwent further enrichment using index PCR. The V(D)J Enriched Library required two rounds of amplification with specific primers. The 5' Gene Expression Library was processed through end repair, addition of a single 'A' base, adapter ligation, and subsequent purification and PCR enrichment. Quantification of the purified libraries was performed using qPCR as outlined in the qPCR Quantification Protocol Guide (KAPA), and quality assessment was conducted using the Agilent Technologies 4200 TapeStation. Finally, sequencing was carried out on the HiSeq platform (Illumina) according to the read length specifications provided in the user guide. Single-cell ATAC Libraries were generated using the Chromium controller in accordance with the 10x Single Cell ATAC protocol (CG000168). Initially, nuclei were isolated and incubated with Transposase in a Transposition Mix. These transposed nuclei were then combined with master mix, Single Cell ATAC gel beads, and Partitioning Oil in a Chip E, where DNA fragments are barcoded in droplets during thermal incubation. The resulting barcoded DNA fragments were pooled and underwent sample index PCR. Quantification of the purified libraries was performed using qPCR as described in the qPCR Quantification Protocol Guide (KAPA), and quality assessment was conducted with the Agilent Technologies 4200 TapeStation. Finally, sequencing was carried out on the HiSeq platform (Illumina) following the read length specifications provided in the user guide.