GSM7993745: Normal1_RNA; Homo sapiens; OTHER
Under sterile conditions, the tissues of ACC1, ACC2, ACC3 and ACC4 were washed twice with pre-cooled RPMI 1640+0.04%BSA medium. The tissues were fully cut into smaller pieces with surgical scissors and put into the freshly conFigd enzymolysis solution with collagenase II in RPMI 1640. The enzymolysis was digested in a constant temperature incubator at 37℃ for 40 min, and mixed inversely every 5-10min. The digested cell suspension was filtered by BD 40μm cell screen for 1 time, centrifuged at 4℃ and 300g for 5min. After the precipitation was suspended with appropriate medium, the same volume of red blood cell lysate (MACS, No. 130-094-183) was added, mixed, left for 10 min at 4℃, cell suspension 300g centrifuged for 5min, and the supernatant was discarded. The precipitation was washed in the medium once, centrifuged at 300g for 5min, and the supernatant was discarded. Follow the MACS Dead Cell Removal Kit (130-090-101) instructions. The cell suspension was added with 100μl magnetic beads, blown and mixed well, incubated at room temperature for 15min and then passed through the column. The eluted cell suspension was centrifuged at 4℃ and 300g for 5min and washed twice, then re-suspended with 100μl medium and Luna was counted. The nuclear suspension preparations experiment of Normal1, Normal2, Normal3, ACC5, ACC6, ACC7, ACC8, ACC9 and ACC10 were conducted on ice. Fresh tissue samples were cut in 1ml nuclear lysate (NST), which was divided into 0.1% NP40, 10mM Tris-HCl, 146mM NaCl, 1mM CaCl2, 21mM MgCl2 and 1U/μl RNase inhibitor. The cracking time was 7min. After the nucleolysis was confirmed by Trypan blue microscopy, 1ml ST Wash buffer (10mM Tris-HCl, 146mM NaCl, 1mM CaCl2, 21mM MgCl2) was added. 0.01% BSA (NEB B9000S) and 40U/mL RNase inhibitor). Filtrate was transferred to a 15mL centrifuge tube through a 40µm cell sieve (BD). The filtrate was rinsed with an appropriate amount of ST Wash buffer and combined with the nuclear filtrate. Centrifuge the horizontal rotor 500g at 4℃ for 5 min. The nucleus was re-suspended with 5ml PBS+1%BSA, and after washing and centrifugation, 100µl PBS+1%BSA was re-suspended. Trypan blue staining, microscopic counting. The single-nuclei suspensions were diluted to 700-1200/µl with PBS+1%BSA. The freshly prepared single-cell suspensions were adjusted to 700-1200 cell/μl, according to 10×Genomics Chromium Next GEM Single Cell 5ʹ Reagent Kits v3.1 (1000020). T Cell V(D)J enrichment was performed in these samples according to the 10×Genomics Chromium™ single-cell V(D)J enrichment kit, human T Cell (1000005) operating instructions, and the library was amplified using Chromium single-cell human TCR amplification kit (1000252). Machine and cDNA libraries were amplified according to the operating instructions of 10×Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (1000268). The constructed library was sequenced using the Illumina Nova 6000 platform in PE150 sequencing platform. 10X 5' scRNA+TCR 10X 3' scRNA