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
V6.5 embryonic stem cells
cell type
Embryonic stem cells
antibody
WCE
treatment
Control

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were crosslinked for 10 min at room temperature by the addition of one-sixth of the volume of 6 % formaldehyde solution (diluted from methanol-free formaldehyde (Thermo Scientific)) to the growth media followed by 5 min quenching with 250 mM glycine. Cells were washed twice with cold PBS, then the supernatant was aspirated and the cell pellet was flash frozen in liquid nitrogen. Frozen crosslinked cells were stored at -80 ºC. Cell pellets were resuspended in ChIP Lysis buffer and ChIP Dilution buffer, treated with MNase for 30 min at -37 ºC followed by quenching with EDTA and EGTA, and further sonicated for 10 cycles at 30 s each with BioRupter. MNase-digested and sonicated lysates were cleared and incubated overnight at 4 ºC with 10 μg DGCR8 or Drosha antibody, and further incubated with pre-washed Dynabeads Protein G (Thermo Scientific) for 2 hrs. Beads were washed two times with sonication buffer, one time with sonication buffer with 500 mM NaCl, one time with LiCl wash buffer and one time with TE with 50 mM NaCl. After resuspension in elution buffer and reversal of crosslink at 65 ºC overnight, RNA and protein were digested using RNase A and Proteinase K, respectively, and DNA was purified with phenol chloroform extraction and ethanol precipitation. After end-repair and A-tailing, ChIP DNA or whole cell extract DNA (50 ng) were ligated to diluted Illumina Adaptor Oligos and size-selected by two step AMPure bead selection (0.9x and 1.8x). After 16 cycle amplification with Phusion HF DNA polymerase, libraries were size-selected with 1.8x AMPure beads.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
53339103
Reads aligned (%)
96.7
Duplicates removed (%)
19.2
Number of peaks
445 (qval < 1E-05)

mm9

Number of total reads
53339103
Reads aligned (%)
96.6
Duplicates removed (%)
19.2
Number of peaks
464 (qval < 1E-05)

Base call quality data from DBCLS SRA