GSM1615104: KO1_AI; Mus musculus; OTHER
Fragmented gDNA samples was labeled in a suspension of azido 1,3-indandione (AI) in 100 mM MES buffer (pH 6.0) for 24 h at 37°C, leading to selective and efficient labeling of 5fC. Click chemistry labeling with DBCO-S-S-Biotin (400 mM) was performed for 2 h at 37°C, followed by purification with QIAGEN PCR purification kit.The Dynabeads MyOne Streptavidin C1 (Invitrogen) was used to pull-down the biotin-labelled DNA with minor modifications on the suggested immobilizing procedure for nucleic acids. In brief, a NaOH-solution washing step (incubated in freshly prepared 0.15 M NaOH at room temperature for 10 min) was added following the canonical washing steps (5 times with 1× binding and washing buffer with 0.1% Tween-20). After that, beads were resuspended and incubated in freshly prepared 50 mM DTT to release the 5fC-containing strand for 2 h at 37 °C. Then the supernatant containing the desired DNA was purified with Micro Bio-Spin P-6 Gel Columns (Bio-Rad) to remove DTT. The fC-CET enriched gDNAs were used directly for library preparation using the TELP protocol. TDT (NEB) enzyme was used to add a poly-dC to allow primer extension of complemwntary strand. MightyAmp DNA Polymerase (TAKARA) was used for one round of on-bead primer extension before PCR amplification. The adaptor-ligated samples were then PCR amplified using NEBNext 2× PCR Master Mix (NEB) and indexed primers (NEB). Libraries were checked using the Agilent 2100 Bioanalyzer DNA 1000 Chip. Two biological replicates of each mESCs were prepared and sequenced, which means that in parallel, two non-enriched input DNAs(Input: preAI), two AI labeled samples (Input: AI) and two pull-down output samples were sequenced simultaneously following the same procedure.
GEO Accession
GSM1615104