Targeted BS-Seq of Zonotrichia albicollis: nestling female nucleus taeniae of the amygdala, white-striped morph
Brains were quickly removed and flash frozen on dry ice, then cryosectioned at 300 m and regions of interest (HYP, POM, and TnA) were micro-dissectied using a 1 mm punch tool following the Palkovits technique (Palkovits et al., 1973). A total of 4 punches were collected from HYP and TnA and 1 punch from POM of each bird 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 beta-mercaptoethanol using a pellet mixer. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) at a of 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. Genomic DNA was bisulfite converted using the EZ Methylation-Lightning Kit following the manufacturers instructions (Zymo, Irvine, CA, USA). Thirteen amplicons were amplified using PCR. All 13 amplicons for each sample were pooled and 5 l of that pool was used for next-generation sequencing. Adapter-ligated libraries were constructed using the 16S Metagenomic Sequencing Library kit (Illumina; San Diego, USA) following the manufacturers instructions. Samples were then run on the Agilent bioanalyzer to confirm successful indexing and quality of total DNA, then pooled for a single sequencing run (PE300) on the Illumina MiSeq at the Emory Integrated Genomics Core.