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).