Illumina NovaSeq 6000 paired end sequencing; ATAC-seq analysis of quiescent human splenic B cell populations
ATAC-seq analysis of quiescent human splenic B cell populations
All spleen in this study were collected from organ donors who died from stroke or head trauma. None of them had lymphoma or autoimmune disease. Immediately after splenectomy, mononuclear cells were isolated from crushed pieces of splenic tissue by ficoll density-gradient centrifugation and stored in liquid nitrogen. After thawing, mononuclear cells were counted and stained with antibodies directed against surface markers. CD21hiCD20hi (CD21high IgG+ memory B cell) and CD21intCD20int memory B cell subsets (CD21int IgG+ memory B cell) were then bulk FACS-sorted (AriaIII) from gated IgG+ switched memory B cells (IgG+CD27+CD20+IgD-) in parallel with naives B cells (CD20+CD38lowCD24intCD27-IgD+CD3-CD14-CD16+). Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) was performed on 50000 cells of each sorted population according to published methods (Buenrostro, Nature Methods, 2013, PMID: 24097267). Libraries were amplified and prepared according to published methods (Buenrostro, Nature Methods, 2013, PMID: 24097267).