Illumina NovaSeq 6000 paired end sequencing; RNA-seq analysis of quiescent human splenic B cell populations
RNA-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+). RNA was extracted from remaining cells using a RNeasy Plus Microkit (Qiagen) and sent for library preparation and sequencing by Integragen (Evry, France). Library preparation was performed by Integragen (Evry, France) using the NEBNext Ultra II Directional RNA kit. Briefly the key stages of this protocol are successively, the purification of PolyA containing mRNA molecules using poly-T oligo attached magnetic beads from 1Î¼g total RNA (with the Magnetic mRNA Isolation Kit from NEB), a fragmentation using divalent cations under elevated temperature to obtain approximately 300bp pieces, double strand cDNA synthesis and finally Illumina adapters ligation and cDNA library amplification by PCR for sequencing.