GSM5597169: C4; Ovis aries; Bisulfite-Seq
Genomic DNA was isolated from thalamus using the Quick DNA midiprep Plus kit (Zymo Research). Degradation of DNA was checked by agarose gel electrophoresis, DNA purity (260/280 ratio) was assessed using NanoDrop spectrophotometer (Thermo Scientific) and the quality and quantity of DNA were determined using and Qubit® 2.0 fluorometer (Life Technologies). Sequencing libraries were constructed for the different genomic DNA samples. Approximately 2.5 µg of genomic DNA spiked with 12.5 ng of lambda DNA were fragmented by sonication to 200-400 bp with Biorupter, followed by end repair and adenylation. These DNA fragments were treated with bisulfite using EZ DNA Methylation-Gold Kit (Zymo Research) followed by size selection and PCR amplification using KAPA HiFi HotStart Uracil + ReadyMix (2×). Library concentration was firstly quantified by Qubit2.0, and then diluted to 1 ng/l before checking insert size on Agilent 2100 and quantified with more accuracy by QPCR (effective concentration of library > 2 nM). Libraries were then sequenced on the Illumina HiSeq Xten platform and 150 bp paried-end reads were generated according to Illumina's protocol.