title: GSM3573220: 10xfemale head 20% Sucrose for 24h 48h Sample 16; Nasonia vitripennis; Bisulfite-Seq
library_construction_protocol: Briefly, tissue was homogenised using a micropestle in 350 µl CTAB (Hexadecyltrimethylammonium Bromide) buffer with subsequent overnight proteinase K digestion (400 µg) at 56 °C. Samples were cooled to room temperature prior to RNase A digestion for 1 hour at 37 °C. A chloroform:isoamyl-alcohol wash (300 µl) was performed twice before ethanol/sodium acetate precipitation of the DNA. DNA purity was analysed using the Nanodrop spectrophotometer with all samples having a 260/280 ratio of ≥1.8. DNA integrity was confirmed using agarose gel electrophoresis. Bisulphite conversion, library preparation and sequencing were carried out by Edinburgh Genomics. Briefly, bisulphite conversion was carried out using the EZ DNA Methylation Kit (Zymo Research, Irvine USA), designed to reduce template degradation during the harsh bisulphite treatment whilst ensuring complete conversion of the DNA. Library preparation was carried out using the TruSeq DNA methylation Kit which takes the single-stranded DNA that results from bisulphite treatment and converts it into an illumina sequencing library. Sequencing of bisulphite-treated DNA was carried out on the Illumina HiSeq 2500 in high output mode with 125bp paired-end reads. Library preparation failed for three of the samples due to low levels of input DNA, leaving only two biological replicates for three out of 18 treatment combinations and a total of 51 Bisulphite-sequenced samples