GSM4879993: D10_M1_TLR; Mus musculus; RNA-Seq
Spleen tissue was harvested from sex- and age-matched C57BL/6, Blimp-1-Venus19 or Prdm1Δex1A mice16 (provided by E. Bikoff, University of Oxford) between 6-12 weeks old that had been maintained in specific-pathogen-free (SPF) conditions. All procedures were performed under the approved UK Home Office project license in line with the Animal (Scientific Procedures) Act 1986 and in accordance with the UK National Cancer Research Institute Guidelines for the welfare of animals. Single cell suspensions were obtained after density centrifugation using lympholyte-M (Cedarlane). The EasySep™ mouse B-cell isolation kit (STEMCELL Technologies) containing anti-CD43 was used to isolate B2 cells. Following isolation, B cells were phenotyped to ensure >95% purity before culture RNA was extracted with TRIzol (Invitrogen) then subjected to DNAseI treatment (DNAfree, Ambion). RNA samples were quantified with a Qubit HS RNA assay kit (Thermo Fisher Scientific) and the quality was assessed by RNA HS assay using the TapeStation system (Agilent). Sequencing libraries were generated by using a TruSeq Stranded Total RNA Human/Mouse/Rat kit (Illumina) according to the manufacturer's protocol. Library performance was checked on the TapeStation followed by picogreen quantification. Equimolar pooling was performed across all the libraries and the final pool was sequenced on a NextSeq 500 platform (Illumina) using 76-bp single-end sequencing.