title: GSM5468469: TKO-L-p0_IAPez; Mus musculus; OTHER
library_construction_protocol: Genomic DNA was isolated from cells through phenol-chloroform extraction 200 ng of genomic DNA was digested with 10 U of BsaWI (NEB), DdeI (NEB), Eco47I (Thermo Fisher Sc.) or MspI (Thermo Fisher Sc.), incubated for 16 h at 60°C (BsaWI) or 37°C (DdeI, Eco47I and MspI) and afterwards heat inactivated. 50 pmol of endonuclease specific hairpin-linker was ligated in a 20 µl reaction at 16 °C for 3 h. Bisulfite conversion was done using Zymo's EZ DNA Methylation-Gold Kit according to manufacturer's instructions. Bisulfite treated Hairpin-DNA was eluted in 20 µl Elution Buffer. PCR was performed in 30 µl reactions with 2 µl of Hairpin-Bisulfite template and 37 PCR cycles using HotFirePol®DNA Polymerase (Solis BioDyne) or HotStarTaq DNA Polymerase (Qiagen). PCR products were purified from a 1.2% agarose gel using Avegene's GenepHlow™ Gel/PCR Kit according to manufacturer's instructions and eluted in 20 µl 0.1x TE buffer. Amplicons were indexed in a 50 µl PCR using specific TruSeq indices and 6 PCR cycles. Index PCRs were purified with 1.1x AMPure XP beads (Beckman Coulter). Amplicons were sequenced on an Illumina MiSeq in a 2x250 bp sequencing mode