GSM1521870: Smolt_Female_0307; Oncorhynchus mykiss; Bisulfite-Seq
DNA was freshly extracted from caudal fin tissue using the Qiagen DNeasy kit according to the manufacturer's suggested protocol. RRBS libraries were constructed using a previously published protocol (Boyle et al. 2012, Genome Biology). Briefly, 100 ng genomic DNA was digested using the restriction enzyme MspI. DNA fragments were end-repaired, A-tailed, and ligated to the methylated Illumina TruSeq adapters. Ten samples were multiplexed per library with each sample ligated to unique barcoded Illumina adapters to distinguish samples. Life history types and sex ratios were primarily equalized across libraries. Bisulfite conversion was conducted using Qiagen's EpiTect Bisulfite kit, following the protocol for formalin-fixed paraffin-embedded samples. After two rounds of bisulfite conversion, the DNA was amplified using Illumina TruSeq primers. The optimal number of PCR cycles, in this case 18, was determined empirically by comparing PCR products on a 5% Metaphor agarose gel for 10, 13, 16, 19, and 22 total cycles. Library size selection was gel-free and occurred by using AMPure XP beads as suggested by Boyle et al. 2012. Library DNA quantity was assessed with the Qubit fluorometer and quality was assessed with a 5% MetaPhor agarose gel (Lonza). All sequencing was performed by the Vincent J. Coates Genomics Sequencing Laboratory at the University of California, Berkeley. After a quantitative PCR quality control check, the Illumina HiSeq 2000 platform was used to sequence 50 bp single-end reads for all 20 samples. The mRRBS protocol was repeated for nine samples with fewer than 10 million reads after quality filtering. Read length was increased for these samples up to 100 bp single-end reads. Two of the smolt (migrant) samples had insufficient reads (<10 million reads) and were removed from subsequent analysis. Therefore, 8 smolts were compared to 10 residents.