epiGBS pool blood Parus major
Each of the eight sequencing libraries contained 36-37 samples: 64 parental samples and 222 nestling samples from 20 cross-foster pairs. We assessed genome-wide DNA methylation levels using epiGBS2 (Gawehns et al., 2022), with some improvements. Sequencing libraries were prepared at the NIOO-KNAW. DNA was isolated from 2 to 5 L blood per sample using the FavorPrep 96-Well Genomic DNA Extraction Kit (Favorgen). 800 ng DNA per sample was digested with MspI. Large fragments were removed using beads (0.8x AMPure XP beads). The fragments were ligated to a unique barcoded adapter combination for each sample within a sequencing library, after which the fragments of all 36 to 37 samples were pooled into one sequencing library. The pooled fragments were exposed to sodium bisulfite, which converts unmethylated cytosines (Cs) into uracils, which will later be amplified as thymines. Bisulfite-PCR amplification was conducted with 15 PCR cycles and the KAPA HIFI Uracil + hotstart ready mix. The  amplified libraries were sequenced on an Illumina HiSeq X (150 bp, paired-end, directional) by Novogene (Novogene (HK) Company Limited, Hong Kong).