title: GSM8017627: OAT, donor 3, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: CD3+ T cells were isolated from PBMCs obtained by FER (n=5), OAT (n=4), and iNOA (n=6) subjects by magnetic beads negative selection (Miltenyi Biotech, Germany).  After generation of nanoliter-scale Gel bead-in-EMulsions (GEMs), GEMs were reverse transcribed in a T100 Thermal Cycler (Bio-Rad, CA, USA) at 53 °C for 45min, followed by 85 °C for 5min, and held at 4 °C. Single-cell droplets were broken, and the single-strand cDNA was isolated and cleaned with Cleanup Mix containing Dynabeads MyOne SILANE (10x Genomics, CA, USA). cDNA was amplified using 13 PCR cycles, and then fragmented, end-repaired, A-tailed and adaptor ligated, with SPRIselect Reagent Kit (Beckman Coulter, CA, USA) with cleanup in between steps according to the manufacturers' instructions. Single-cell libraries were generated through the Chromium Single-Cell Controller using the Chromium Single Cell 3′ Reagent kit v.3.1 according to the manufacturers' instructions. (10x Genomics, CA, USA). For each donor, samples were loaded to obtain a target cell recovery of 8000 cells. The concentration of the scRNA-seq libraries was determined using Qubit dsDNA HS Assay kit (Invitrogen, MA, USA) at Qubit 3.0, and size distribution was assessed using an Agilent D1000 TapeStation system (Agilent, CA, USA). Libraries were sequenced on an Illumina NovaSeq6000 instruments with a paired-end 150-bp (performed by GENEWIZ, Germany).