GSM9177758: PBMCs, RRMS, S14; Homo sapiens; RNA-Seq
PBMCs were isolated from whole blood by Ficoll density-gradient centrifugation, washed in PBS + 0.04% BSA, and kept on ice. Nuclei were prepared according to the 10x Genomics demonstrated protocol CG000365 (Nuclei Isolation for Single Cell RNA Sequencing): brief lysis on ice, quenching, gentle trituration, filtration through a 40-µm strainer, and washes in 10x Nuclei Buffer. Intact nuclei were counted and resuspended in 10x Nuclei Buffer at the target concentration for Chromium loading. Single-nucleus 3′ gene expression libraries were generated on the Chromium Next GEM platform using the Single Cell 3′ Reagent Kit v3.1 (Dual Index) following the manufacturer's instructions. Nuclei were encapsulated in GEMs with gel beads, reverse transcription was performed within GEMs to barcode cDNA with a cell barcode and UMI, GEMs were broken, cDNA was purified and amplified, and sequencing libraries were constructed by fragmentation, end-repair, A-tailing, adaptor ligation, and sample index PCR. Libraries were quantified and sequenced on an Illumina platform (paired-end).