GSM8625330: 0_Gy_plus_4PBA; Mus musculus; Bisulfite-Seq
Genomic DNA was extracted with QIAamp® DNA Micro Kit (Qiagen). Genomic DNA is incubated with 200U MspI (New England Biolabs) at 37°C for 2h. Digested product is then end-repaired, A-tailed and ligated to methylated Illumina adaptors with the NEB Next Ultra II kit (New England Biolabs) following provider recommandations. After ligation step, DNA fragments are purified and size-selected with a two step SPRI beads purification in order to select fragments from 40bp to 400bp. Clean-up ligated DNA is eluted in Low TE buffer. Bisulfite conversion is performed following provider recommandations (Zymo EZ-lightning kit). Converted product is finally amplified by PCR using polymerase Kapa HiFi Hotstart U+ Ready mix 2X in a final volume of 50 μL. Indexed libraries with Illumina primers are PCR amplified with 13 cycles following provider incubation protocol. PCR product is then purified by 1.2X SPRI beads clean-up and controlled by capillary electrophoresis on Fragment Analyzer. Diagenode Premium RRBS kit