title: Mouse Marginal Zone B-cell with/without Tfh RNASeq data analysis
design: Mouse Marginal Zone B-cell with/without Tfh RNASeq data analysis
library_construction_protocol: C57BL6 adult mouse sorted Marginal Bcells in spleen at 16-wk old. Euthanized with CO2 and then opened abdomen to get spleen. Spleen then smashed and filtered after obtaining a suspension with isolated spleen cells. Then B cells were pre-enriched from the suspension using Stem Cell B cell isolation kit. Cells were stained for B220, CD21 and CD23 and sorted in Aria III sorter (Cd23low Cd21hi population). 16wks C57BL6 mouse RNA was isolated from sorted MZB Micro RNA Plus (Quiagen) from reconstituted lethally irradiated LDLr-/-: Rag2-/- (mice that do not have T and B cells) with a BM containing 100% CD4Cre/+; Bcl6flox/flox (No Tfh) or a mixed chimaera containing 80% CD4Cre/+; Bcl6flox/flox + 20% CD4+/+; Bcl6flox/flox (WT). Align to GRCm39 reference genome using star (version 2.6.1d) RNA was isolated from sorted MZB Micro RNA Plus (Quiagen) from reconstituted lethally irradiated LDLr-/-: Rag2-/- (mice that do not have T and B cells) with a BM containing 100% CD4Cre/+; Bcl6flox/flox (No Tfh) or a mixed chimaera containing 80% CD4Cre/+; Bcl6flox/flox + 20% CD4+/+; Bcl6flox/flox (WT). The quality of the RNA extracted was checked using a Bioanalyser RNA Pico kit (Agilent) with RIN values > 7. RNA (2.5 ng) was whole transcriptome-amplified using the Ovation RNA-seq System V2 (NuGEN). Prepare the RNA-seq library, the amplified cDNA (2Î¼g per sample) was fragmented to 200 bp using a Bioruptor Sonicator (Diagenode), and barcode ligation and end repair were performed using the Ovation Rapid DR Library System (NuGEN).