Summary: RNA-seq in the LXS RI Panel Following an Intraperitoneal Injection of Saline
+ +RNA-seq-derived gene expression was determined from the LXS recombinant inbred strains and the two parental strains (ILS/Ibg and ISS/Ibg) that had been treated with normal saline (ip) 8 hours before being sacrificed. (This is a companion to a similar dataset in which the same strains were treated with 5 g/kg ethanol [20% v/v in normal saline, ip] and sacrificed at 8 hours). Breeders were obtained from the Jackson Laboratory and experimental mice were bred in-house at the University of Colorado Anschutz Medical Campus. All samples were from whole brain (minus cerebellum and olfactory bulbs) of male mice at an average age of 80 days (SEM: +/- 0.3; range: 58-106; median: 82). The rationale for the dosing and the implicit overall rationale for this experiment can be found in Radcliffe et al. (2006); Radcliffe et al. (2013); Darlington et al. (2013); and Bennett et al. (2015).
+ +A total of 396 mice representing 43 LXS RI strains and the ILS and ISS were used. Following total RNA isolation, an equal amount of RNA from three mice of each strain was quantitatively pooled and then the samples were enriched for poly-A RNA using the Dynabeads mRNA Purification kit (Invitrogen) as directed by the manufacturer. Paired-end (2x100, expected size of 300 bp), strand-specific, cluster-ready libraries were prepared from the poly-A enriched RNA using the ScriptSeq RNA-Seq Library Preparation Kit v2 (Illumina). Three libraries per strain were prepared, 132 in total. Due to poor quality or other technical difficulties, 9 libraries were eliminated leaving a total of 41 strains (including ILS and ISS) comprised of 36 strains with n=3, 3 strains with n=2 and 2 strains with n=1. Tophat (Trapnell et al., 2009) was used to map reads to RI-specific genomes; i.e., the ILS and ISS were sequenced (see Bennett et al., 2015) and with the use of genotype data from the LXS (see Saba et al., 2011), a genome was created for each RI strain. Mapped reads were then quantified at the gene level using HTSeq (Anders et al., 2015) with Ensembl full gene annotations and then converted to FPKM using the formula FPKM=fragments/kb exon/million mapped reads/2 (note that this assumes that both reads of a pair were successfully mapped; for a small percentage of the reads this was not the case and these were treated separately and added in). FPKM values were converted to log2 (FPKM+1) which gives a value of 0 for FPKM=0, 1 for FPKM=1, 2 for FPKM=3, 3 for FPKM=7, 4 for FPKM=15, and so on.
-- cgit v1.2.3