Rna 6000 pico bioanalyzer chips

To quantify patterns of gene expression, we first isolated RNA from the liver samples collected during this study. A 15mg portion of each sample was homogenized using a PowerLyzer^® 24 with ceramic beads (MO BIO Laboratories, Inc.), after which RNA was extracted from the homogenized sample using the UltraClean^® Tissue and Cells RNA isolation kit (MO BIO Laboratories, Inc.). The concentration of each extract was determined using a NanoDrop (ThermoFisher); typically, extractions yielded ~ 200ng/μL of RNA. The quality of each extract was assessed using RNA 6000 Pico BioAnalyzer chips (Agilent); the mean RNA integrity number (RIN) score for our samples was 8 (range = 7.1–8.9). RNA extractions were then further purified using the DNase Max^® kit (MO BIO) or, for samples requiring additional concentration, RNeasy spin columns (Qiagen). Finally, samples were diluted to a standard concentration of 80 ng/μl in 25 μl of RNAase‐free water (total = 2000 ng RNA) for library preparation.

To quantify transcriptomic patterns of genetic diversity, we first isolated RNA from a subset of the liver samples collected during this study (four males and four females per species per sampling location), resulting in extracts from 64 individuals. For each sample, a 15 mg portion of liver was homogenized using a PowerLyzer^® 24 equipped with ceramic beads (MO BIO Laboratories, Inc.), after which RNA was extracted from the homogenized sample using the UltraClean^® Tissue and Cells RNA isolation kit (MO BIO Laboratories, Inc.). The concentration of each extract was determined using a NanoDrop spectrophotometer (ThermoFisher); typically, extractions yielded ~ 200 ng/μL of RNA. The quality of each extract was assessed using RNA 6000 Pico BioAnalyzer chips (Agilent); the mean RNA integrity number (RIN) score for our samples was 8 (range = 7.1 to 8.9). RNA extractions were then further purified using the DNase Max^® kit (MO BIO) or—for samples requiring additional concentration—RNeasy spin columns (Qiagen). Finally, samples were diluted to a standard concentration of 80 ng/μL in 25 μL of RNAase-free water (total = 2000 ng RNA) for library preparation.