RNA-Seq of Calidris pugnax: satellite male, caudal nidopallium
Total RNA from seven microdissected brain areas and four organ samples was extracted using the RNeasy Mini Kit (Qiagen) according to manufacturers instructions. One modification was that we homogenized testicular tissue and filtered it through QIAshredder columns before initiating the RNA isolation protocol. RNA concentration and RNA to protein ratios were quantified (A260/A280) on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). RNA integrity numbers (RINs) were measured with RNA Nano Chips on an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA samples were diluted and concentrations re-measured with the Qubit RNA BR Assay Kit (Thermo Fisher Scientific) on a Qubit Fluorometer. After shipment to the sequencing provider, total RNA quality for all samples was re-assessed using a Fragment Analyzer 5200 (Agilent). Polyadenylated RNA enrichment was performed using the NEBNext Poly (A) mRNA Magnetic Isolation Module Kit (New England BioLabs). RNA libraries were synthesized using the NEBNext Ultra II Directional RNA Library Preparation Kit for Illumina (New England Biolabs). 250 ng of total RNA from each sample was subjected to 100 bp paired-end sequencing on an Illumina NovaSeq 6000 (12 PCR cycles) according to Illumina protocols. Conversion of binary base call (BCL) sequence files and demultiplexing were performed using bcl2fastq2 v2.2.0.