GSM5176052: Tet_TKO_168h-2i-BS; Mus musculus; Bisulfite-Seq
Genomic DNA was prepared using QIAamp DNA Micro Kit or AllPrep DNA/RNA mini kit (QIAGEN). 1.2 μg DNA is divided equally into three 0.5ml reaction tubes and digested in a 20 μl reaction using 20 U of HaeIII (NEB), AluI (NEB) and 10 U HpyCH4V (NEB), respectively. The reactions are incubated overnight for a minimum of 12 h at 37 °C. Restriction enzymes are inactivated at 80 °C for 30 min. The reactions are pooled and subjected to a ligation step. During this process, hairpin linker and sequencing adapter are introduced to the opposed ends of each restriction fragment. For this, 200 mM biotin labeled hairpin linker and 100 mM sequencing adapter are added to the digested DNA, incubatedwith 1 mM ATP and 2000 U T4 DNA Ligase (NEB) for 16 h at 16 °C. The reaction is purified using AMPureXP beadsfollowed by enrichment for hairpin containing fragments with streptavidin beads. The library is then subjected to BS/oxBS work-flow of the TrueMethyl kit from CEGX according tomanufacturer's instructions. Amplification of the library was done with HotStarTaq® polymerase (Qiagen) and sequencing was performed on an Illumina HiSeq 2500 platform in a 150 bp paired-end sequencing mode.