GSM5284399: GC B cells from RagC +/+ mice [YRC00684]; Mus musculus; RNA-Seq
Germinal Centre B cells (B220+, CD95+,CD38−) were sorted from RagCQ119L/+ and RagC+/+ splenocytes after 12 d of SRBC immunization in a BD FACSAria Ilu (Becton Dickinson) and InFlux (Cytopeia-Becton Dickinson) cell sorters. Total RNA was extracted using TRIzol and PicoPure RNA Isolation kit (Arcturus, #12204-01) following the manufacturer's instructions. Total RNA samples were processed into cDNA sequencing libraries with the "QuantSeq 3' mRNA-Seq Library Prep Kit (FWD) for Illumina" (Lexogen, Cat.No. 015). Library generation is initiated by reverse transcription with oligodT priming, followed by a random-primed second strand synthesis. A UMI Second Strand Synthesis module was used whereby random primers featured a 6 nt long Unique Molecular Identifier 5' tag. Primers from both steps contain Illumina-compatible sequences. Libraries are completed by PCR, and sequenced on an Illumina instrument (see below) by following manufacturer's protocols. {This library preparation kit generates directional libraries stranded in the sense orientation: the read1 (the only read in single read format) has the sense orientation}