title: epiGBS pool blood Parus major
design: The sequencing libraries each contained DNA extracted from blood from 36 Parus major nestlings (180 in total). DNA methylation was assessed using the reduced-representation bisulfite sequencing method epiGBS2 (Gawehns et al., 2022). EpiGBS2 library preparations were conducted at the NIOO-KNAW following the laboratory protocol described in the Supporting Information of Sepers et al. (2023). 800 ng DNA per sample was extracted using the FavorPrep 96-Well Genomic DNA Extraction Kit (Favorgen) and digested with the restriction enzyme MspI (NEB). The fragments were ligated to a unique barcode combination for each sample and the fragments from 36 samples were pooled (i.e. multiplexed). To be able to differentiate methylated cytosines from unmethylated cytosines, unmethylated cytosines were converted to thymines using sodium bisulfite. Sequencing was performed by Novogene (Novogene (HK) Company Limited, Hong Kong) on an Illumina HiSeq X (150 bp from paired-end, directional reads). PhiX DNA was spiked-in (12%).