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
Mouse Embryonic Stem Cells (mESHA-Tet1)
strain
C57BL/6N
genotype
HA-Tet1
agent
mock treated
antibody
none

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-Seq of endogenous HA-Tet1 in mouse embryonic stem cells (mESHA-Tet1) was performed on cells grown on 0.1% gelatin-coated plates in DMEM 20% FCS and 0.4% leukemia inhibitory factor. Cells were permeabilized for 15min in cold PBS with 0.05% Tween 20 at 4°C, washed with cold PBS and were incubated with RNase H1 0.2 U/ul (NEB) and Nuclease S1 0.05 U/ul (Fermentas) or mock treated for 20 min at RT. The cells were then washed and crosslinked in PBS with 2mM disuccinimidyl glutarate (DSG) for 45 min. The cells were washed and crosslinked again with 0.7% formaldehyde for 15 min and quenched with 0.5 M glycine for 5 min. Finally the cells were washed with PBS, lysed and the chromatin was sonicated using Bioruptor Pico (Diagenode) for 10 cycles of 30 sec on, 30 sec off to yield 250 bp average size fragments. The sonicated chromatin was immunoprecipitated with HA antibody (Abcam ab9111) at a ratio of 1.5 ug HA antibody per 20 ug chromatin. For Inputs, 5% of the chromatin from each replicate was collected and pooled into one Input control sample per condition. The subsequent ChIP procedures were performed as described in Arab et al. 2014 (doi:10.1016/j.molcel.2014.06.031). ChIP-seq libraries were prepared using NuGEN Ovation Ultralow System V2 following the manufacturer’s recommendations. The libraries were profiled on a 2100 Bioanalyzer (Agilent Technologies) and quantified with a Qubit 2.0 Fluorometer (Life Technologies). The samples were pooled in equimolar ratio and sequenced on NextSeq 500 (Illumina) in paired-end mode for 42 cycles (PE42) plus 7 cycles for the index read.

Sequencing Platform

instrument_model
NextSeq 500

mm10

Number of total reads
65561194
Reads aligned (%)
96.5
Duplicates removed (%)
9.3
Number of peaks
361 (qval < 1E-05)

mm9

Number of total reads
65561194
Reads aligned (%)
96.4
Duplicates removed (%)
10.1
Number of peaks
377 (qval < 1E-05)

Base call quality data from DBCLS SRA