title: GSM6721771: Con3; Homo sapiens; RNA-Seq
library_construction_protocol: 2ml peripheral blood from each patient or healthy volunteer in EDTA anticoagulant tube was diluted with 2ml 1×PBS. Equal volume Ficoll lymphocyte separation solution (solarbio, P4350) was added into 50ml centrifuge tube. The diluted blood was slowly spread onto the lymphocyte separation solution. Centrifugation was conducted at 20 ℃ with the horizontal rotor at 2000rpm for 20min, with break set to 0. The intermediate white membrane layer cells (PBMCs) were carefully extracted into 15ml centrifuge tube. The white membrane layer cells were washed with 10ml 1×PBS and were centrifuged with the horizontal rotor at 300g for 10min. The supernatant was discarded, and the cells were suspended with 5ml 1×PBS. The suspension was centrifuged at 300g for 10min and washed twice. The supernatant was discarded again, and the cells were suspended with 1ml RPMI 1640 medium (Conring,Art.10-040-CVR) added with 0.04%BSA. Luna cell counter or Trypan blue staining was used to count single cell suspension concentration and cell viability. Single-cell RNA sequencing libraries were constructed by using Chromium Single cell 3' Reagent v2 Kits in accordance with the manufacturer's protocol. PBMC suspensions were loaded on the Chromium Single Cell Controller Instrument (10×Genomics) with partitioning oil, reverse transcription (RT) reagents, and a collection of gel beads containing 3,500,000 unique 10 × barcodes. Single cell gel beads were generated in emulsions (GEMs). Transcription reactions were performed to generate barcoded full-length cDNA. Emulsions were disrupted by the recovery agent and barcoded-cDNA was purified with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). cDNAs were amplified with appropriate cycles (98°C for 3 minutes; cycled 12 times: 98°C for 15 second, 63°C for 20 second, and 72°C for 1 minute; 72°C for 1 minute; end at 4°C) and cleaned up with the SPRIselect Reagent Kit (Beckman Coulter, USA). The amplified cDNA received fragmentation, end-repair, polyA-tailing, index adaptor ligation. PCR amplification was conducted by sample indexing primers (98°C for 45 sec; 14-16 cycles of 98°C for 20 sec, 54°C for 30 sec, 72°C for 20 sec; 72°C for 1 min; end at 4°C) and double-sided size selected with SPRIselect beads. High-throughput sequencing was performed on the Illumina sequencing platform (Illumina Nova 6000) and 150 bp paired-end reads were generated.