GSM9659385: hPCO_D6 (scATAC-seq); Homo sapiens; ATAC-seq
Nuclei isolation from snap-frozen tissue was performed with the Nuclei Isolation method according to the manufacturer's instructions(10Xgenomics，CG000212).In brief, frozen tissue samples(30-50mg/sample) are homogenized with a pestle in 1.5ml tube with 500 ul chilled 0.1XLysis Buffer，and incubate on ice for 15 min. Pass the suspension through a 70 µm and a 40 µm Flowmi Cell Strainer. Centrifuge collection tube for 3 min at 500 rcf at 4°C. Carefully discard supernatant using a pipette without disturbing nuclei pellet. Resuspend nuclei pellet in 0.2 ml hilled Diluted Nuclei Buffer Determine the nuclei concentration using a Countess II FL Automated Cell Counter. The isolated nuclei are then loaded directly into compatible10XChromium NextGEM SingleCell ATAC ReagentKitsv2 assays. Following nuclei isolation, samples were sequenced by 10x Chromium single-cell platform (CG000496 Chromium NextGEM SingleCell ATAC ReagentKits v2 UserGuide RevB). In detail, Nuclei suspensions are incubated in a Transposition Mix that includes a Transposase. The Transposase enters the nuclei and preferentially fragments the DNA in open regions of the chromatin. Simultaneously, adapter sequences are added to the ends of the DNA fragments.Then ,Gel Beads-in-emulsion (GEMs) generation and barcoding was performed. Post GEM incubation cleanup and DNA amplification were conducted. Finally, the scATAC library construction was performed following the manufacturer's instructions. Libraries are generated and sequenced on NovaSeq6000 (Illumina).