GSM9058623: p52ki3Nai_22; Mus musculus; RNA-Seq
RNA was extracted from FACS-sorted naïve, activated,  pre-GC and GC B cells and stored at -80°C. The total RNA was extracted using Arcturus PicoPure RNA Isolation kit (Thermo Fisher Scientific) according to the manufacturer's protocol. RNA was assessed using either RiboGreen (Thermo Fisher Scientific) or Agilent Bioanalyser. cDNA libraries were prepared using 0.5 ng of total RNA, or half of the RNA sample where concentration was too low to be accurately quantified. The SMARTSeq v2 protocol was used with 17 PCR cycles and the following modifications: 1. Addition of 20 µM TSO; 2. Use of 200 pg cDNA with 1/5 reaction of Illumina Nextera XT kit. The length distribution of the cDNA libraries was monitored using a DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip. All samples were subjected to an indexed paired-end sequencing run of 2x151 cycles on an Illumina Novaseq 6000 targeting at least 13 million read-pairs/sample.