GSM5457171: MultiplexedPBMC; Homo sapiens; ATAC-seq
Cryopreserved PBMCs isolated from three healthy unrelated donors were obtained from Hemacare (Los Angeles, CA, USA). PBMCs were thawed and cultured in RPMI 1640 with GlutaMax and HEPES (ThermoFisher, cat#72400047) supplemented with 10% FBS and 1% pen/strep overnight on ultra low-attachment 10cm dishes (Corning, cat#3262). Following overnight culture, suspension PBMCs were transferred to a 50mL conical tube and placed on ice. Adherent PBMCs were lifted using TrypLE and pooled with the suspension PBMCs. The cells were then washed twice with ice-cold PBS before nuclei isolation according to the “Nuclei Isolation for Single Cell ATAC Sequencing” protocol from 10x Genomics. Isolated nuclei were then resuspended in ice-cold 10x Genomics diluted nuclei buffer and counted. 15,000 nuclei from each PBMC donor were then subjected to transposition according to the “Chromium Next GEM Single Cell ATAC Reagent Kits v1.1” protocol from 10x Genomics. After transposition, nuclei from each donor were pooled, pelleted, resuspended in 10x Genomics ATAC Buffer B, and counted prior to microfluidic capture (targeting ~10,000 recovered nuclei). Next-generation sequencing library preparation was then performed according to supplier recommendations before sequencing on a single lane of a Illumina NovaSeq SP100 flow cell.