White-throated sparrow (Zonotrichia albicollis) liver and pectoralis flight muscle transcriptomic changes in preparation for migration
Approximately 50 mg of pectoralis muscle and liver tissue were homogenized using beadmill homogenization (NextAdvance, Troy, NY; speed 8, time 3 for liver 4 for pectoralis, 4C) in 1 mL of TriZol (Invitrogen, Carlsbad, CA) and RNA was separated into an aqueous phase using a chloroform ethanol procedure. Total RNA was purified using a PureLink RNA kit (Invitrogen) and DNase treated using TURBO I DNase (Invitrogen). Samples were quantified using Qubit (Invitrogen) and RNA quality (RIN > 8.2) was confirmed using an Agilent 2100 Bioanalyzer. Libraries were prepared using an NEB Ultra II Directional kit (New England Biolabs, Ipswich, MA) with poly(A) mRNA magnetic isolation and dual-index primers according to the manufacturers protocol. We confirmed library quality and fragment sizes using the Bioanalyzer 7500 kit for dsDNA (Agilent, Santa Clara, CA) prior to pooling 96 samples (including additional samples from a separate study) to a final library concentration of 50 M. We sequenced the pooled libraries using the NovaSeq 6000 platform (Illumina, San Diego, CA) multiplexed on a single S4 flow cell for paired-end 150 bp sequencing (GENEWIZ, South Plainfield, NJ).