RNAseq analysis of distinct small intestinal cell populations isolated from Lgr5-EGFP C57BL/6 mice treated with NRG1

Purpose: The goal of the study was to investigate the effect of Neuregulin 1 (NRG1) on intestinal crypt-based columnar stem cells, their progenitors and differentiated cells. Methods: Lgr5-EGFP C57BL/6 male mice (11-15 week-old, littermates) were injected with two intra-peritoneal doses of NRG1 daily (7.5 µg every 12 hours) or vehicle control (PBS) for 5 days (n=3). On day 6, small intestinal tissues were collected and several intestinal cell populations isolated by FACS based on Lgr5-GFP and CD24 expression [CBC stem cells (Lgr5high-CD24low), their immediate progenitors (Lgr5med-CD24low), subsequent progenitors (Lgr5low-CD24low) and differentiated cells (double negative)]. RNA was then extracted for each cell population and RNA sequencing analysis conducted. Overall design: mRNA profiles of intestinal CBC stem cells, progenitor cells and differentiated cells isolated from NRG1-treated or PBS-treated mice were generated by RNA sequencing, in triplicate. Libraries of each biological replicate were prepared with 15ng of RNA using the Nugen Ovation RNA-Seq system V2 kit followed by Nugen Ovation Ultralow System V2 kit. High throughput sequencing was performed with HiSeq3000 with ~30 M reads targeted reads (single end 76nt length) per sample.