ImmGen 11-cell set basic RNASeq project

Full depth directional RNA sequencing was performed on the core ImmGen populations to generate reference datasets for the tissues from 5 week-old C57BL/6J (Jackson Laboratory) males and females, double-sorted by flow cytometry, per ImmGen cell preparation SOP. RNA was prepared from 100,000 cells (final sort collected directly in Trizol). Total RNA was extracted from each sample following ImmGen RNA extraction SOP. PolyA+ RNA was isolated by bead capture (PrepX PolyA mRNA Isolation Kit, Wafergen Biosystems), following the manufacturer''s instructions. Then mRNA libraries were prepared with the PrepX RNA-Seq Library Preparation Kit (Integenex). For first strand synthesis, mRNA was fragmented and ligated to 3'' and 5'' adapters before reverse transcription. Sample libraries then underwent bead clean-up, 2nd strand synthesis and PCR amplification before final sequencing. Libraries were analyzed on an Illumina HiSeq2500 sequencer. The read depths for the 11-cell set populations range from around 8 to 25 million.