title: Day 6 RNA
organism: Homo sapiens
description: Protocols: Peripheral blood mononuclear cells (PBMCs) from 3 healthy controls were obtained by venepuncture, separated by Ficoll density gradient centrifugation and cryopreserved in FCS with 10% DMSO at -80oC for a week. PBMCs were thawed, and total B cells were obtained by negative selection using the EasySep™ Human B Cell Isolation Kit (StemCell Technologies, #17954). Total B cells were then stained for further flow cytometry sorting with anti-CD20, anti-CD27, anti-IgD. Viability staining using LIVE/DEAD™ Fixable Near IR Viability Kit (Invitrogen, #L34975) was performed together with the surface staining. Naïve B cells (CD27-IgD+) were sorted by flow cytometry using a BD FACSAria™ Fusion and BD FACSDiva software version 9.4, and collected at 4oC in recovery media (50% heat-inactivated FBS + 50% RPMI 1640 (Sigma-Aldrich, #R7388). Naïve B cells at D0 were washed in PBS with 5% FBS and run on the 10X as described in the single-cell methodology below. In parallel, the rest of the sorted naïve B cells, 250k cells per well, were then cultured in vitro under IgG polarising class-switch consisting of complete RPMI (10% heat-inactivated foetal bovine serum and 1% penicillin/streptomycin) + 1µg/ml anti-IgM (JacksonImmunoResearch, #309-006-043) + 1µg/ml CD40L (Enzo Life Sciences, #ALX-522-110-C010) and 50ng/ml IFNγ (Bio-Techne, #285-IF-100/CF) at 37oC and 5% CO2 for 3 or 6 days. IgG polarising class-switch medium was refreshed on day 2 by gently centrifuging the cells (300g for 10min) and adding fresh media with the above mentioned factors. Cells were counted and run on a Chromium 10X controller using 5' v2 chemistry (10X Genomics) in one lane with an expected recovery rate of 5,000 cells per lane, according to the manufacturer's instructions. For each timepoint samples from each healthy control were multiplexed separately using TotalSeq hashtags1, 2 and 3 (Biolegend #394661, #394663, #394665) and anti-CD27 Total Seq (Biolegend, #302853) for 30 min in PBS with 5% FCS supplement. Pooled and hashtagged libraries were generated according to 10X Genomics instructions.