Bulk RNAseq of human GPR183+CD27+ B cells
Live CD19+GPR183+CD27+ B cells were isolated from PBMCs on a FACSAria sorter (BD Biosciences) and RNA was extracted using PicoPure RNA Isolation Kit (Thermo Fisher) according to manufacturers instructions. RNA quality control and sequencing were performed by UCL Genomics (UCLG). RNA integrity was confirmed using the Agilent 4200 Tapestation (Standard Total RNA assay). Samples were processed using the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (JSLE and HC samples). The cDNA library was prepared from mRNA that was isolated from total RNA by use of Oligo dT beads. Full length xGen adaptors (IDT), containing two unique 8bp sample-specific indexes, a unique molecular identifier and a T overhang, were ligated to the A-tailed cDNA and enriched with limited cycle PCR (16 PCR cycles). Samples were sequenced on a NovaSeq (Illumina) at 300pM using a 101bp paired read run with corresponding 8bp dual sample index and 8bp unique molecular index reads. Run data were demultiplexed and converted to fastq files using BCL Convert Software v3.7.5 (Illumina). Transcript abundance was estimated using a custom Galaxy pipeline developed by UCLG to produce a raw count table. Data were adapted and quality trimmed using fastp before being aligned to the human genome (UCSC hg38) with RNA-STAR. Aligned reads were then UMI deduplicated using JE-Suite. FeatureCounts was used to estimate reads per transcript by counting uniquely mapped reads that fall within coding or UTR regions of the genome. All sequence and annotation data were obtained from the Illumina iGenomes repository.