Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Unclassified
Antigen
Unclassified

Cell type

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

Attributes by original data submitter

Sample

source_name
murine embryonic stem cells
cell line
E14 with Dnmt1 KO generated using CRISPR/Cas9

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed, washed with ice-cold PBS, and pelleted. Cell pellets were lysed through sonication in a Bioruptor (UCD-200) and supernatants were saved. 30 μL of chromatin was stored overnight at 4°C for input samples while the remainder of the chromatin was combined with antibody coupled protein A magnetic beads (NEB) and incubated at 4°C overnight with constant rotation. Dnmt1 antibody (Bethyl) coupled protein A magnetic beads (NEB) were blocked with 5 mg/ml BSA overnight at 4°C, prior to incubation with sheared chromatin. Magnetic beads were washed and material was eluted at 65°C on a thermomixer. Eluted material was transferred to a new microfuge tube, combined and incubated at 65°C overnight to reverse crosslinking. Input DNA was diluted with 170 μl elution buffer and treated similarly. Samples were treated with RNaseA/T1 (Ambion), proteinase K (Ambion), and then PCI extracted. Ethanol precipitated ChIP-encriched DNA was then used for library construction. 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-) 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
35455414
Reads aligned (%)
83.8
Duplicates removed (%)
17.7
Number of peaks
342 (qval < 1E-05)

mm9

Number of total reads
35455414
Reads aligned (%)
83.7
Duplicates removed (%)
17.8
Number of peaks
298 (qval < 1E-05)

Base call quality data from DBCLS SRA