Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
TET2

Cell type

Cell type Class
Kidney
Cell type
293
Primary Tissue
Kidney
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
HEK293T_HA_Tet2_ChIP
cell line
HEK293T
transfected with
HA-Tet2
chip antibody
HA beads

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
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

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
63749317
Reads aligned (%)
76.2
Duplicates removed (%)
10.9
Number of peaks
18956 (qval < 1E-05)

hg38

Number of total reads
63749317
Reads aligned (%)
77.3
Duplicates removed (%)
10.0
Number of peaks
19298 (qval < 1E-05)

Base call quality data from DBCLS SRA