title: RNA-seq of Zonotrichia albicollis hypothalamus
design: Brains were quickly removed and flash frozen on dry ice. Brains were cryosectioned and regions of interest (HYP and TnA) regions were micro-dissectied 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. Stranded libraries were prepped  using 250 ng total RNA.