Experimental_sample_stickleback_TG33_RS71
Library preparation followed the Smallwood and Kelsey reduced representation bisulfite sequencing (RRBS) protocol (Smallwood & Kelsey in Genomic Imprinting: Methods and Protocols, N. Engel, 686 Ed., pp. 187-197). 100-250 ng purified DNA was digested with the methylation-insensitive MspI restriction enzyme. DNA end-repair and A-tailing was conducted and un-tailed CEGX spike-in controls (Cambridge Epigenetix) were added. After adapter ligation, bisulfite conversion was conducted using the EZ-96 DNA Methylation-Gold Kit (Zymo Research) according to the manufacturers protocol. PCR amplification with 19 cycles was performed. Quality control of purified PCR products was performed on a 2200 TapeStation System (Agilent) and high-quality libraries were pooled and diversified with 15% PhiX. Single-end sequencing with 100 bp read length was conducted on a HiSeq 2500 sequencer (Illumina).