title: GSM1937520: HEK293T_Tet2+MBD3L2_RRBS; Homo sapiens; Bisulfite-Seq
library_construction_protocol: For ChIP-seq, cells were fixed with DSG (Disuccinimidyl glutarate) followed by formaldehyde. Chromatin was incubated with anti-HA antibody (Cell Signaling; C29F4) for overnight followed by incubation with Protein A magnetic beads (Millipore). ChIP DNA was reverse-crosslinked and purified with MinElute PCR purification kit (Qiagen); For hMeDIP-seq, genomic DNA was sonicated to <500 bp by Bioruptor sonicator (Diagenode; Belgium) and quantified using the NANODROP2000c (Thermo) according to the manufacturer’s manual. The sonicated DNA fragments (1ug of each sample) were end-repaired using the End-It DNA End Repair Kit (EPICENTRE Biotechnologies) according to the manufacturer’s instructions, followed by treatment with Klenow fragment 3’-5’exo (NEB) and dATP to generate a protruding 3’A for ligation with the adaptor containing a specific barcode sequence. The barcode sequence (four-base index: TTAGGC, GATCAG, AGTTCC, GTCCGC) within the adaptor. Then equal amount of bar-code tagged gDNA from Control, MBD3L2, Tet2, Tet2+MBD3L2 were mixed together in one tube and 10% DNA was taken as Input. The mixed DNA was then incubated with 5hmC antibody (Active Motif) for overnight followed by incubation with Protein A/G beads. The 5hmC binding DNA fragments were purified with MinElute PCR purification kit (Qiagen); For RRBS, 500 ng of genomic DNA was digested with the methylation insensitive restriction digest enzyme Msp1 (NEB), which cleaves DNA at CCGG sites creating fragments high in CpG content (Gu et al., 2011). The DNA was purified using the QIAquick PCR Purification Kit (Qiagen). For ChIP-seq, library were performed using NEB Next ChIP-Seq Library Prep Master Mix Set (NEB), following manufacturer instructions; For hMeDIP-seq, Library were performed using NEBNext ChIP-Seq Library Prep Master Mix Set (NEB), following manufacturer instructions; For RRBS, samples were end-repaired and A-tailed using a Klenow fragment (New England Biolabs, Inc). TruSeq™ adaptors (Illumina, Inc., San Diego, CA) were ligated to the modified DNA ends using T4 DNA ligase (New England Biolabs, Inc.). We excised the gel ranged from 150-400bp and purified the gel with the QIAquick Gel Extraction Kit (Qiagen). The sample was bisulfite modified and PCR amplified to enrich for fragments containing high CpG content. Samples were submitted for sequencing on the Illumina HiSeq™ instrument
attributes.tag: GEO Accession
attributes.value: GSM1937520