title: GSM8626432: HEK293T cells, Ac_MeDIP; Homo sapiens; MeDIP-Seq
library_construction_protocol: MeDIP-Seq: After 72 h of transfection, cells over-expressing Lc, Ac, Gg, Hs and Ev were harvested for genomic DNA isolation using phenol-chloroform-isoamyl alcohol extraction method. Genomic DNA was sonicated to obtain 700-1000 bp fragments. 1µg fragment DNA was end repaired using NEBNext Ultra II End Repair/dA-Tailing Module (NEB #​​E7546). PCR adapters from Oxford Nanopore Technologies (ONT #SQK-PSK004) were ligated to end repaired DNA using NEB Blunt/TA Ligase Master Mix (NEB #M0367). 1x MeDIP master mix (10 mM Sodium Phosphate Buffer, 0.14 M NaCl and 0.05% TritonX-100) was added to adapter ligated DNA which was then denatured at 95℃ for 5 mins followed by snap chilling on ice. An antibody cocktail against 5-methylcytosine (EMD Millipore #MABE146, Sigma #SAB2702243 and Novus Biologicals #NBP2-42813) was added to snap-chilled DNA and was incubated at 4℃ overnight. Protein G Plus/Protein A Agarose beads were added to the mix followed by incubation at room temperature for 2 h. Then beads were collected by centrifugation in a separate tube and were washed thrice with 1x MeDIP master mix buffer and were subjected to Proteinase K (Sigma #P2308) digestion at 56℃ for 2 h. The slurry was centrifuged and immunoprecipitated DNA in the supernatant was collected into new tubes. This DNA was subjected to 18 cycles of PCR using whole genome primers (ONT #SQK-PSK004) and LongAmp Hot Start Taq 2X Master Mix (NEB #M0533). MeDIP-Seq: Library preparation was done using NEBNext® UltraTM II DNA Library Prep Kit for Illumina® (NEB #E7645, #E7103) as per manufacturer's instruction. DNA was quantified using Qubit and quality assessment was done using TapeStation. DNA was sequenced on Illumina HiSeq 2500 platform