GSM8328105: Blood,excercised,cell830,wk12; Homo sapiens; RNA-Seq
Seven to ten million cryopreserved PBMCs were sorted for E2-specific B cells as described previously. S figure 4, in the article indicates the time points selected for flow analysis and transcriptomics/repertoire analysis. In brief, PBMCs were stained with an in-house developed rE2 cocktail tetramer (Gt1a [H77] and Gt3a [UKN3A13.6]) as well as anti-CD19, anti-CD20, anti-CD10, anti-CD38, anti-IgD, anti-IgG, anti-CD27,anti-CD21 and anti-CD95 antibodies, and single cell sorted using flow cytometry. Population phenotypes were analysed with FlowJo version 10 software (Tree Star Inc.) (See CTAT Table for antibody details and see S Figure 3 for gating strategy and how gates were set for CD95 positivity based on FMO). E2+ B cells were sorted into single wells and single cell RNA sequencing was performed following the smartseq2 protocol as previously described. BCR sequences were reconstructed using VDJpuzzle 2.0. Identification of B cell clonotypes and SHM across infections was done using Immcantation framework.  Paired-end reads from Smart-seq2 were aligned with STAR (version 2.7.1a) using the GRCh38.p13 human reference genome. Transcripts per Million (TPM) were quantified using RSEM (version 1.2.28) using rsem-calculate-expression command on HPC clusters.