GSM8641134: 3340Tp; Mus musculus; RNA-Seq
Negative selection of spleen B-cells was performed with the Pan B Cells isolation kit of Miltenyi® (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, cells were labeled with the anti-CD19-BV510 monoclonal antibody (BD Biosciences, Franklin Lakes, NJ) for cell sorting. CD19pos TOM+ and TOM- B-cells from TOM-AID mice and CD19pos TOM-/YFP-, TOM+/YFP- and TOM+/YFP+ from REL-AID mice were sorted on a Facs Aria III Cell Sorter (BD Biosciences). Total RNA was extracted with the RNeasy Kit (Qiagen, Les Ulis, France) following the manufacturer's protocol and quantified with the NanoDrop One (Thermo Scientific™, Walham, MA). Purification of polyA mRNA was done from 100ng total RNA with the Magnetic mRNA Isolation Kit (New England Biolabs, Ipswich, MA). Libraries were prepared with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina protocol according supplier recommendations (New England Biolabs). Fragmentation using divalent cations under elevated temperature to obtain approximately 300bp pieces, double strand cDNA synthesis and finally Illumina adapters ligation and cDNA library amplification by PCR for sequencing were performed.