WGBS of epiRIL-368 grown at cold temperature
Libraries were prepared for using a KAPA high throughout Library Prep Kit -with amendments (Part No: KK8234). This kit has been optimised for 1g of input DNA with a size selection using Beckman Coulter XP beads (Part No: A63880). The DNA was QCd with a High Sensitivity Qubit assay (part No: Q32854) and 1 g in 55 l of each sample was taken forward for processing. The DNA was sheared using the Covaris LE220 sonicator (Covaris) to an average size of 350bp. Methylated barcoded adapters (NEXTFlex bisulfite barcodes (BiooScientific _ 511913)) are then added to the treated ends of sheared DNA. Bisulfite Conversion of DNA library was performed using EZ DNA Methylation-Gold Kit. Treating DNA with bisulfite chemically modifies non-methylated cytosines into uracil, methylated cytosines remain unchanged. After PCR enrichment, the insert size of the libraries is verified by running an aliquot of the DNA library on a PerkinElmer GX using the High Sensitivity DNA chip (Part No: 5067-4626) and the concentration determined by using a High Sensitivity Qubit assay. A 12-plex equimolar pool was then prepared ready for q-PCR and sequencing on the HiSeq 4000 using v1 chemistry and 150bp paired-end reads over 2 lanes. The total number of raw reads generated in the WGBS-seq data was ~ 23 M per biological replicate.