Bulk BCR-seq of mouse lymph nodes NP-OVA immunization IL-21 treated
About 4x105 purified BGC cells were processed for RNA extraction using Qiagen RNeasy Mini kit (Qiagen). The BCR libraries were generated using NEB Next Immune Sequencing Kit (Mouse) (NEB, MA, USA) according to the manufacturers protocol. In brief, the cDNA was synthesized using oligo d(T) primers together with a template-switch adapter with UMIs, and purified with streptavidin magnetic beads. Then the cDNA of BCR was amplified using a set of C-region-specific primers together with sample index primers, followed by the second PCR using the universal primers and another index primers. Before the second PCR, the cycle number was determined by a qPCR using the same primers and templates as per the manual. PCR products (~650bp) were purified by streptavidin magnetic beads. Libraries were pair-end (400+100bp) sequenced on a MiSeq (Illumina) in the Institute of Molecular Biology, UQ.