GSM6500937: B3; Homo sapiens; RNA-Seq
2 ml PBMCs from gout patients were obtained in EDTA-anticoagulated and the whole blood was diluted with 1×PBS. Equal volume Ficoll lymphocyte separation solution (solarbio, P4350) was added into the 50ml centrifuge tube, and the diluted blood was slowly spread onto the lymphocyte separation solution. Centrifugation was conducted at 20 ℃ with the horizontal rotor at 2000rpm for 20min, and break was set to 0. Carefully drain the intermediate white membrane (PBMC) cells into the new 15ml centrifuge tube. White membrane layer cells were washed with 10ml 1×PBS. 300g, centrifuged for 10min. The supernatant was discarded, then the cells were suspended with 5ml 1×PBS, centrifuged at 300g for 10min and washed twice, then the supernatant was discarded, and the cells were suspended with 1ml RPMI 1640 medium (Conring, Art. 10-040-CVR) supplemented 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-seq libraries were generated by using Chromium Single cell 3' Reagent v2 Kits according to the manufacturer's protocol. PBMC suspensions were loaded on the Chromium Single Cell Controller Instrument (10×Genomics) to with partitioning oil, reverse transcription (RT) reagents, and a collection of gel beads that contained 3,500,000 unique 10× barcodes. generate single cell gel beads in emulsions (GEMs). Following GEMs generation, reverse transcription reactions were engaged barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and barcoded-cDNA was purified with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). Whereafter, cDNAs were amplified with appropriate cycles (98°C for 3 min; cycled 12 times: 98°C for 15 sec, 63°C for 20 sec, and 72°C for 1 min; 72°C for 1 min; end at 4°C) and cleaned up with the SPRIselect Reagent Kit (Beckman Coulter, USA). Subsequently, the amplified cDNA was fragmented, end-repaired, polyA-tailed, index adaptor ligated. PCR amplified with 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. Then these libraries were sequenced on the Illumina sequencing platform (Illumina novaseq 6000) and 150 bp paired-end reads were generated.