UCAMC LXS Whole Brain Ethanol RNA Sequence (Feb16) FPKM **

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Anders S, Pyl PT, Huber W (2015) HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31:166-169.
Bennett B, Larson C, Richmond PA, Odell AT, Saba LM, Tabakoff B, Dowell R, Radcliffe RA (2015) Quantitative Trait Locus Mapping of Acute Functional Tolerance in the LXS Recombinant Inbred Strains. Alcoholism: Clinical and Experimental Research 39:611-620. Darlington TM, Ehringer MA, Larson C, Phang TL, Radcliffe RA (2013) Transcriptome analysis of Inbred Long Sleep and Inbred Short Sleep mice. Genes Brain Behav 12:263-274.
Radcliffe RA, Floyd KL, Lee MJ (2006) Rapid ethanol tolerance mediated by adaptations in acute tolerance in inbred mouse strains. Pharmacol Biochem Behav 84:524-534.
Radcliffe RA, Larson C, Bennett B (2013) Genetic studies of acute tolerance, rapid tolerance, and drinking in the dark in the LXS recombinant inbred strains. Alcohol Clin Exp Res 37:2019-2028.
Saba LM, Bennett B, Hoffman PL, Barcomb K, Ishii T, Kechris K, Tabakoff B (2011) A systems genetic analysis of alcohol drinking by mice, rats and men: influence of brain GABAergic transmission. Neuropharmacology 60:1269-1280.
Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105-1111.

Specifics of this data set

Summary: RNA-seq in the LXS RI Panel Following an Intraperitoneal Injection of Ethanol

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 5 g/kg ethanol (20% v/v in normal saline, ip) 8 hours before being sacrificed. (This is a companion to a similar dataset in which the same strains were treated with 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: +/- 0.4; range: 56-106; median: 81). 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 44 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, 5 libraries were eliminated leaving a total of 44 strains (including ILS and ISS) comprised of 40 strains with n=3, 3 strains with n=2 and 1 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.