GSM9468896: PBMC, pediatric MCD, n=4; Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood by density-gradient centrifugation using Ficoll-Paque. Whole blood was diluted 1:1 with DPBS and carefully layered onto Ficoll (Ficoll: diluted blood = 15 mL:25 mL in 50 mL tubes, or 5 mL:10 mL in 15 mL tubes). Samples were centrifuged at 2000 rpm for 20 min at 20°C. The PBMC layer at the plasma–Ficoll interface was collected and transferred to pre-warmed DPBS, followed by two washes. Cells were counted, resuspended in cryopreservation medium, aliquoted into cryovials, and cryopreserved using a controlled-rate freezing container before storage at liquid nitrogen. For library preparation, cryopreserved PBMCs were thawed and processed according to the manufacturer's guidelines Libraries were prepared according to the manufacturer's guidelines  (Chromium Next GEM Single Cell 3' HT, 10x Genomics). Thawed cells were resuspended in the supplied master mix and co-loaded with gel beads and partitioning oil into the microfluidic chip to form GEMs. Each GEM served as an isolated reaction chamber where cells were lysed and polyadenylated transcripts were reverse-transcribed into cDNA. The resulting cDNA molecules were tagged with a 10x cell barcode and UMI, and also contained the Illumina Read 1 priming sequence. Barcoded cDNA was then pooled, the emulsion was removed, and cDNA was cleaned up using Silane DynaBeads prior to PCR amplification. Size selection of amplified cDNA was performed with SPRIselect reagent, and the selected fragments were converted into sequencing libraries by adding the Illumina Read 2 priming sequence, P5/P7 flow-cell adapters, and a sample index for multiplexed paired-end sequencing.