title: RNA-seq of Zonotrichia albicollis: adult female hypothalamus (white morph, rep3)
design: Brains were quickly removed and flash frozen on dry ice. Brains were cryosectioned at 300um thickness and hypothalamus region was micro dissectied from frozen setions using a 1mm punch tool. A total of 4 punches were collected from each bird and region and were pooled for RNA extraction. RNA was extracted using the Qiagen Allprep DNA/RNA micro kit with modification, as follows. Punches were homogenized in RLT plus + -mercaptoethanol using a pellet mixer. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) pH 7.5 was added to each sample. Samples were further homogenized by passing them through a Qiashredder column (Qiagen). Phases were separated by spinning for 15 min at 4C, and the aqueous phase was collected and further processed according to the manufacturers protocol. Libraries were prepped using Illumina TruSeq RNA Sample Preparation Kit v2. Libraries were constructed using 250 ng total RNA. The libraries were then sequenced on the HiSeq 4000 at 150 bp paired-end reads to ~40 million reads per sample.