title: RNA-Seq of Homo sapiens: CD4+ B cells high respondant to flu shot
design: RNA was  quantified and integrity was analyzed on Agilent Tape Station. Samples with  RNA integration number of greater than 8 were selected for library  preparation. Poly-A enrichment of mRNA was performed from 15ug of total RNA  (Dyna beads mRNA direct Micro Kit). All samples were spiked in with ERCC  spike in mix (Life Technologies) before poly-A enrichment for performance  assessment of libraries. 100ng of poly-A enriched RNA was fragmented using  RNase III and purified using magnetic bead clean up module (RNA Seq V2 kit,  Life Technologies). Size distribution of the fragmented RNA was assessed on  Agilent Tape Station using RNA HS screen tape. 50ng of fragmented poly A  enriched RNA was used to prepare whole transcriptome library using RNA seq V2  kit( Life Technologies ) following manufacturer recommended protocols . Yield  and size distribution of the library  was assessed on Agilent Tape Station using D1000 screen tape. Barcoded  libraries were equally pooled and amplified onto Ion Sphere Particles (ISPs)  supplied by Ion Pi HiQ OT2 kit (Life Technologies). ISPs loaded with  libraries were sequenced on Ion Torrent Proton sequencer using Ion PI chip  V3. Two samples were pooled together and sequenced in one chip (Ion PI HiQ  sequencing kit).