GSM9369279: 10x5_PBMC_09252020; Homo sapiens; RNA-Seq
CD45+ immune cells were isolated from the kidney biopsy tissue and the matched PBMC sample by flow sorting. Sorted CD45+ cells were processed using the Chromium Next GEM Single Cell 5' Kit (10x Genomics). A single-cell suspension, reverse transcription reagents, gel beads containing barcoded oligonucleotides, and oil were loaded to a Chromium controller (10x Genomics) to generate Gel Beads-In-Emulsions (GEMs). Within each GEM, the encapsulated cell underwent lysis and reverse transcription, producing full-length cDNA uniquely barcoded to its cell of origin. Next, the GEMs were broken, and cDNA from each cell was pooled. The cDNA underwent purification using Dynabeads MyOne Silane Beads and was then PCR-amplified. The amplified product was optimally fragmented, followed by end-repair, A-tailing, and adaptor ligation. Final libraries were generated by PCR amplification. Library concentrations were quantified using the KAPA Library Quantification Kit (Roche) and sequenced individually on an Illumina NovaSeq 6000 platform.