GSM6171460: ATF7IP-KO_WGBS; Mus musculus; Bisulfite-Seq
Genomic DNA extraction, followed by phenol/chloroform extraction and ethanol precipitation. RNaseA treatment was performed for 30 min at 37C 10 µg of genomic DNA was sonicated to a length of approximately 400-500 base pairs. For each sample, 2 µg of sheared genomic DNA was mixed with 10 ng of equimolar pooled sonicated methylated phage T7 and unmethylated phage Lambda DNA. Adapter-ligation was carried out with the NEBNext Ultra II kit (NEB E7645L) using methylated adaptors (NEB E7535S), prior to bisulfite conversion using the Qiagen Epitect bisulfite conversion kit, according to the manufacturer's instructions. After conversion, libraries were amplified for 10 cycles using the Pfu TurboCx Hotstart DNA polymerase (Agilent) and the NEB dual index primers (NEB E7600S). PCR reactions were run with the following parameters:  95°C for 2 min, 98°C for 30 s, followed by 10 cycles of 98°C for 15 s, 65°C for 30 s and 72°C for 3 min, ending with 5 min at 72°C. The PCR reactions were cleaned up using 1.2 x AMPure XP beads (Beckman Coulter) and eluted in 20 µl EB buffer (Qiagen). Library quality was checked on an Agilent TapeStation and sequencing was done on an Illumina NovaSeq machine.