Germinal center B-cell immunoglobulin sequences in NP-immunized mice- NP3
Bonnie JHD-/- MRL/MpJ-Faslp strain female mice (16), 6-10 week old, were immunized with NP-CGG in alum adjuvant. 28 days after the immunization the animals were sacrificed and splenocytes harvested. Germinal center cells (live, B220+, NIP+, CD95+, CD38-) were FACS-sorted from 4 mice; 64000, 87000, 120000, and 107000 cells, respectively. After sorting, cells were spun down, the RNA isolated using the RNeasy Mini kit (Qiagen) per the manufacturer protocol and stored at -80C. 250 ng of RNA was reverse-transcribed into cDNA using a biotinylated oligo dT primer. An adaptor sequence was added to the 3' end of all cDNA, which contains a universal priming site and a 17- nucleotide single-molecule barcode (UID). Products were purified using streptavidin-coated magnetic beads followed by PCR reactions as described in Supplemental Information. Final products were purified, quantified with Agilent Tapestation and pooled in equimolar proportions, followed by high-throughput 325+275 base-pair paired-end sequencing on the Illumina MiSeq platform according to manufacturer recommendation.