GSM4474213: cg1_cre_GCBcells_rep3; Mus musculus; RNA-Seq
Total RNA was isolated from sorted, viable GC (B220+, GL7+, Fas+) B cells using Direct-zolTM RNA MicroPrep kit (catalog # R2061) as recommended by the manufactuer. Briefly, an equal volume of absolute ethanol was added into each sample lysated in Trizol. Samples were mixed, transferred into Zymo-Spin IC Colomns in Collection Tubes, centrifuged and washed. Samples were then treated with DNase I (included in kit) and washed again. DNase/RNase-Free Water (included in kit) was added to elute RNA. Libraries were prepared using the Takara SMART-Seq v4 Ultra Low Input RNA kit (catalog # 634891), as recommended by the manufacturer. An oligo-dT primed capture of total message RNA was used to produce full length transcripts from very low inputs of total RNA (10 ng total RNA input per sample). Samples were indexed using unique dual indices. All 12 libraries were sequenced on a single lane (12-plex) of a  NextSeq 550 instrument on High Output mode, generating at least 30M 100nt single-end reads per sample.