Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Mbd2

Cell type

Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA

Attributes by original data submitter

Sample

source_name
Untagged, Mbd2FLAG, ChIP, murine embryonic stem cells
cell line
E14
cell type
ESC
genotype/variation
control
chip antibody
anti-Flag M2 (Sigma, catalog# F1804, lot# 080M6035)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed, washed with PBS, and pelleted. After resuspention and incubation with Lysis buffers 1 (50 mM HEPES KOH pH 7.6, 140 mM NaC, 1 mM EDTA, 10% (w/v) glycerol, 0.5% NP-40, 0.25% Triton X100) l and 2, pellets were sheared in Lysis buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for sonication in a Bioruptor. Sheared chromatin was incubated at 4°C overnight with protein G magnetic beads (NEB) pre-blocked and coupled with anti-Flag M2 (Sigma F1804). Magnetic beads were washed, material was eluted at 65°C on a thermomixer, and the eluent was incubated at 65°C overnight to reverse crosslinking. Input DNA was diluted with 170 μL elution buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM EDTA, 1% SDS) and treated similarly. Samples were treated with RNaseA/T1 (Ambion) followed by proteinase K (Ambion) and then PCI extracted. Ethanol precipitated ChIP-enriched DNA was then used for library construction or as a template for qPCR reactions. Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (exo minus) and dATP to yield a protruding 3- 'A' base for ligation of barcoded adapters which have a single 'T' base overhang at the 3’ end. DNA purification on Zymo Research PCR purification columns (Zymo, Irvine, CA) was performed following each enzyme reaction. The adaptor-ligated material was then PCR amplified with KAPA-HiFi polymerase using 16 cycles of PCR before size selection of 200-350 bp fragments on a 1% agarose gel. Libraries with different barcodes were pooled together and single-end sequencing was performed on an Illumina HiSeq2000.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
9736253
Reads aligned (%)
96.5
Duplicates removed (%)
11.5
Number of peaks
323 (qval < 1E-05)

mm9

Number of total reads
9736253
Reads aligned (%)
96.3
Duplicates removed (%)
11.8
Number of peaks
323 (qval < 1E-05)

Base call quality data from DBCLS SRA