Brains were dissected from 12 male and 12 female zebrafish and were halved through the sagittal plane. 2 male and 2 female RRBS library was prepared with each library containing a pool of six halved zebrafish brain.  A total of four RRBS libraries were prepared from 2 pooled male brain and 2 pooled female brain library each containing 6 halved zebrafish brain. Genomic DNA was digested with MspI (New England Biolabs, Ipswich, MA) followed by end repair and addition of 30 A overhangs. Methylated adaptors (Illumina, San Diego, CA) with a 30 T overhang were then ligated with the generated fragments. Following adaptor ligation, DNA fragments ranging from 40 to 220 bp (preligation size) were cut from a 3% (w/v) NuSieve GTG agarose gel (Lonza, Basel, Switzerland) and subsequently bisulphite modified using the EZ DNA methylation kit (Zymo Research, Irvine, CA). The final library was amplified by PCR. The resulting library was sequenced on an Illumina platform with a single-ended, 49bp run.
GEO Accession
GSM1456411