GSM8863835: BRCA1-mutant peripheral blood; Homo sapiens; RNA-Seq
The solid tissues (including tumor and adjacent tissues) were processed to prepare single-cell suspensions. The tissue samples were cut into small pieces, and the cells were washed twice with precooled RPMI 1640 + 0.04% BSA medium under aseptic conditions. The tissues were cut into small pieces of approximately 0.5 mm3 using surgical scissors, placed in freshly prepared hydrolase and digested at a constant temperature of 37°C for 60 minutes, mixing every 10 minutes. The tissue was filtered 2 times using a cell sieve and centrifuged at 300 x g for 5 minutes at 4°C. After resuspending the sediment with an appropriate amount of medium, an equal volume of erythrocyte lysis buffer (MACS, catalog number: 130-094-183) was added. After mixing, the cells were incubated at 4°C for 10 minutes and centrifuged at 300 x g for 5 minutes, and the number of viable cells, as determined by Trypan blue staining, was determined with a hemocytometer. Human fresh peripheral blood was collected in an anticoagulant tube and diluted with PBS at 1:1. Then, the sample was added to the Ficoll separation medium (Merck) and centrifugated at 750 x g for 20 minutes. After centrifugation, a white-grayish layer consisting of mononuclear cells will be found on top of the Ficoll-Paque. Carefully collected the mononuclear cells using a Pasteur pipette and transferred them to a new centrifuge tube. Washed the cells by centrifuging the new tube for 10 minutes at 300 x g. The supernatant was discarded and resuspended in RPMI-1640 supplemented with 0.04% BSA. The number of viable cells, as determined by Trypan blue staining, was determined with a hemocytometer. The cell concentration of the freshly prepared single cell suspension was adjusted to 700-1200 cell/μl. The cDNA library amplification was performed according to the operating instructions of 10× Genomics Chromium Next GEM Single Cell 5ʹ Reagent Kits v2.0 (PN-1000263). DNA library construction was performed using the 10x Genomics Chromium Single Cell 5' Kit (PN-1000190) following the manufacturer's guidelines. Purified cDNA libraries were sequenced on an Illumina platform (Illumina Nova 6000). For T-cell receptor (TCR), T cell V(D)J enrichment was performed using the 10× Genomics single cell V(D)J Enrichment Kit (TCR, PN-1000005) according to the human T cell operating instructions. The TCR library was amplified using the Chromium Single Cell TCR amplification Kit (TCR, PN-1000252), and the experimental operation was performed according to the product instructions.