Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Embryo
Cell type
Neural Crest
MeSH Description
Neuroectodermal cells of the neural crest. They differentiate into various cell types during EMBRYOGENESIS including craniofacial MESENCHYME; ENDOCRINE CELLS; MELANOCYTES and PERIPHERAL NERVOUS SYSTEM.

Attributes by original data submitter

Sample

source_name
Cranial neural crest cells
cell type
Cranial neural crest cells
cell subpopulation
Md
age
E10.5
genotype
WT
chip antibody
none
library protocol
NEBNextUltra-Illumina

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For the single ChIP-Seq experiments: Embryonic tissues were micro-dissected at E8.5, E10.5 or E11.5, incubated in trypsin 0.5%/EDTA 1X at 37 °C for 10 minutes, rinsed (DMEM 1X/ FBS 10%, followed by PBS 1X), cross-linked with 1% formaldehyde in PBS 1X for 10 minutes at room temperature (RT) and quenched with 125 mM glycine for 5 minutes at RT. Cranial neural crest cells (CNCCs) were pelleted by centrifugation (2000 rpm, 10 minutes, 4°C) and rinsed three times in ice-cold PBS. CNCCs were collected by FACS and pelleted by centrifugation (2000 rpm, 10 minutes, 4°C). 1 millions of FACS-isolated CNCCs were used for each single ChIp-Seq experiments. CNCCs were incubated in 1 ml of Cell Lysis Buffer (0.5% NP40, 85 mM KCl, 5 mM PIPES pH8) for 10 min on ice. Nuclei were isolated by spinning the lysed CNCCs for 5 minutes at 2500 g, at 4°C. The supernatant was discarded and cell nuclei were re-suspended in Sonication Buffer (50 mM Tris HCl pH8, 10 mM EDTA, 1% SDS, 1X Protease Inhibitor Cocktail (PIC - Complete-EDTA free, Roche)). After 15 min of lysis on a tube shaker at 4°C, the DNA was sonicated using the Covaris machine (settings: cycles per burst 200, duty cycle 10%, peak incidence power 175 Watts for 2400 sec.) to obtain a mean DNA fragment size of 250 bp. After centrifugation (10 minutes, 10 000 rpm, 4°C), the supernatant was transferred in a new tube, diluted 10X with RIPA buffer (10 mM Tris-HCl pH7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X100, 0.1% SDS, 0,1% Na-deoxycholate, 1 mM PMSF, 20 mM NaB, 1X PIC) and incubated over-night at 4°C with 5 μl of antibody. The next day, 50 μl of protein G coupled to magnetic beads (Dynabeads Protein G, Invitrogen) were added and the incubation continued for 2 hours. The beads were then washed 3 times with RIPA buffer and once with TE buffer (10Mm Tris-HCl pH 8.0, 1mM EDTA). The protein-DNA complexes were eluted from the beads with 150 μl of Elution Buffer (20 mM Tris-HCl pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 50 mg/mL proteinase K) at 68 °C for 2 hours, to reverse cross-links and digest proteins. DNA was purified using MinElute PCR purification kit (Qiagen). ChIP libraries were prepared using bar-coded adapters according to standard Illumina library preparation protocols, following the manufactorer’s instructions for the NEBNext® Ultra™ DNA Library Prep Kit for Illumina®, or the TruSeq® ChIP sample preparation for Illumina with and without gel-size selection (for the protocol used, see “library protocol” annotation of each sample). The Quality of the libraries and size distribution was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies). One to four samples with different barcodes were mixed at equimolar ratios per pool. Sequencing was performed on an Illumina HiSeq 2500 machine (50 bp read length, single-end) according to Illumina standards.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

mm10

Number of total reads
106903310
Reads aligned (%)
96.8
Duplicates removed (%)
19.1
Number of peaks
649 (qval < 1E-05)

mm9

Number of total reads
106903310
Reads aligned (%)
96.6
Duplicates removed (%)
19.0
Number of peaks
804 (qval < 1E-05)

Base call quality data from DBCLS SRA