Gene expression analysis
Immediately following blood collection, a subset (n=13) of females was euthanized using isoflurane overdose, followed by rapid decapitation. The sample size for gene expression analyses was small to limit the number of euthanized wild songbirds. The brain was dissected and reserved for another study. The skull with the embedded pituitary gland was flash-frozen on pulverized dry ice and transferred to a -80 °C freezer within 6 hours. Pituitaries were then rapidly dissected from frozen skulls and returned to -80 °C storage.
For RNA-extraction, the tissue was suspended in 500ul Tri-Reagent (Molecular Research Center, Cincinnati, OH, USA) and homogenized. RNA was then extracted using the manufacturer’s protocols, treated with DNase I (New England Biolabs, Ipswich, MA, USA) and purified using QIAGEN RNeasy (Valencia, CA, USA) mini kit.
RNAseq libraries were constructed at the DNA Services laboratory of the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign using the TruSeq Stranded RNA Sample Preparation Kit (Illumina San Diego, CA). Briefly, the total RNA was quantitated by Qubit (Life Technologies, Grand Island, NY), then PolyA+ RNA was selected from 1µg of total RNA per sample. PolyA+ RNA was fragmented for 4 minutes at 94C, then first-strand cDNA was synthesized with a random hexamer and SuperScript II (Life Technologies). Double stranded DNA was blunt-ended, 3’-end A-tailed and ligated to unique dual-indexed adaptors. The adaptor-ligated double-stranded cDNA was amplified by PCR for 10 cycles with the Kapa HiFi polymerase (Kapa Biosystems, Woburn, MA). The final libraries were quantitated on Qubit and the average size determined on the AATI Fragment Analyzer (Advanced Analytics, Ames, IA) and diluted to 5nM final concentration. The 5nM dilution was further quantitated by qPCR on a BioRad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc. CA).
The final stranded RNASeq library pool consisting of 13 libraries was sequenced on 1 lane of an Illumina NovaSeq 6000 SP flowcell as paired-reads with 150nt length. The run generated .bcl files which were converted into adaptor-trimmed demultiplexed fastq files using bcl2fastq v2.20 Conversion Software (Illumina, CA).