Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

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

Attributes by original data submitter

Sample

source_name
ES cells_input
strain
C57BL/6J x Cast/EiJ
cell type
hybrid ES cells

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP assays were performed using formaldehyde cross-linked chromatin isolated from ten 10 cm dishes of hybrid ES cells grown to ~70% confluency. Crosslinking was performed by adding formaldehyde to cell media in the dish to a final concentration of 1% and incubation at room temperature for 10 minutes. The reactions were stopped with glycine to a final concentration of 125 mM. The cells were washed in PBS and re-suspended in cell lysis buffer (0.25 % Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH 8.0) to isolate nuclei. Chromatin was extracted in nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5 % SDS) and sheared to an average size of 200-500 bp using Bioruptor sonicator. Chromatin from ~5x106 cells was diluted 5 fold with IP buffer (1.1 % TX-100, 1.2 mM EDTA, 16.7 mM Tris pH 8.1, 167 mM NaCl) and pre-cleared with 10 µg non-immune rabbit IgG and 50 µl of protein A magnetic beads for 3 hours at 4˚C on a rotating wheel. Anti-Zfp57 antibody (4 µg, Abcam ab45341) was added to pre-cleared chromatin and incubated overnight at 4˚C on a rotating wheel. Chromatin was precipitated with protein magnetic A beads (25 µl slurry in X-ChIP) for further 3 hours at 4 oC with rotation. The beads were washed in each of the following: buffer 1 (1% Triton X-100, 0.1% SDS, 2 mM EDTA, 150 mM NaCl and 20 mM Tris pH 8), buffer 2 (1% Triton X-100, 0.1% SDS, 2 mM EDTA, 500 mM NaCl and 20 mM Tris pH 8), buffer 3 (0.25 M LiCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 10 mM Tris pH 8) and twice with TE buffer (10 mM Tris pH 8, 1 mM EDTA). The bound chromatin was eluted into elution buffer (1% SDS, 0.1 M NaHCO3), crosslinks reversed and protein digested. DNA was extracted by phenol/chloroform and ethanol precipitated in the presence of 10 µg of glycogen. One tenth of ChIP reactions was used for qPCR and SNP-Pyrosequencing analysis to control for ChIP efficiency. The remainder was used for Illumina library preparation. Sequencing libraries were prepared from ~10ng of ChIP and 1 µg of input DNA using NEBnext® kit (E6260, NEB) according to manufacturer's protocol. The PCR amplified library was purified using Agencourt AMPure magnetic beads and gel size selected for 150-400 bp. Each ChIP and input libraries were sequenced from a single end to a length of 40nt on individual lanes of an Illumina Genome Analyzer IIx.

Sequencing Platform

instrument_model
Illumina Genome Analyzer IIx

mm10

Number of total reads
31025973
Reads aligned (%)
95.9
Duplicates removed (%)
11.1
Number of peaks
707 (qval < 1E-05)

mm9

Number of total reads
31025973
Reads aligned (%)
95.6
Duplicates removed (%)
11.1
Number of peaks
787 (qval < 1E-05)

Base call quality data from DBCLS SRA