Transcriptomic Profiling of Peromyscus Tissues

Liver and testis tissues were obtained (and frozen immediately in liquid nitrogen) from scrotal males of four species (P. attwateri, P. boylii, P. leucopus, and P. maniculatus) during the summer of 2012, 2013, or 2014 and archived at the Natural Science Research Laboratory, Museum of Texas Tech University (table 1). Tissues were separately homogenized and RNA extracted using the TRIzol reagent (ThermoFisher) following the manufacturer’s recommended protocol. RNA quality was quantified using a Bioanalyzer System (Agilent Genomics) with a minimum of 7.5 for the RNA Integrity Number score. Sequencing libraries were generated from 500 ng of RNA from each sample using the NEB NEXT library prep kit (New England Biolabs, Beverly, MA). Indexed libraries were pooled in equimolar concentrations and paired-end, 100-bp reads were sequenced from the cDNA libraries using an Illumina HiSeq 2000. Illumina paired-end reads were clipped, trimmed, and orphans were sorted using Trimmomatic v0.27 (Bolger et al. 2014 (link)). To count the number of pairs and improper or orphaned read alignments, reads were aligned using the program, Bowtie2 v2.3.4 (Langmead and Salzberg 2012 (link)). In total, eight transcriptomes were generated, two for each individual, representing the liver and testis samples.