Antigen

Antigen Class

Unclassified

Antigen

Unclassified

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.

Sample

source_name

Cranial neural crest cells

cell type

Cranial neural crest cells

cell subpopulation

Md

age

E10.5

genotype

WT

chip antibody

H3K27me3 (Millipore, Cat. # 07-449) and H3K4me2 (Millipore, Cat. # 07-030)

library protocol

NEBNextUltra-Illumina

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

For sequential H3K27me3/H3K4me2 ChIP-Seq experiments, around 7 million of non-FACS isolated cells were used to perform the first ChIP as described above, up to the washing of beads with TE buffer, using several tubes. Denaturing conditions (DTT, high salt and detergent) and heat were used to release the chromatin from the antibody coated magnetic beads. Right after removal of the TE buffer, the DNA/H3K27me3-antibody/protein-G complexes were re-suspended in 25 ul of DTT 100 mM (5 minutes at RT). Then, 25 ul of Chromatin Release Buffer (500 mM NaCl, 2% SDS, 2% sodium Deoxycholate, PIC 2X) were added to each tube, samples were mixed thoroughly and incubated at 37°C for 30 min. After the release incubation, beads were removed and tubes were pooled, diluted 4 times in IP dilution buffer (10 mM Tris-HCl ph7.5, 140 mM NaCl, 0,1% Na-deoxycholate, 1% Triton X100, 1 mM EDTA, 1X PIC), and concentrated using a 50Kda cutoff Centricon (Amicon). The collected material was then diluted 16 times in IP dilution buffer and incubated over-night at 4°C with 5 μl of anti-H3K4me2 (Millipore). 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. The protein-DNA complexes were eluted from the beads with 150 μl of Elution Buffer at 68 °C for 2 hours. 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

66262977

Reads aligned (%)

73.9

Duplicates removed (%)

78.9

Number of peaks

11121 (qval < 1E-05)

mm9

Number of total reads

66262977

Reads aligned (%)

73.8

Duplicates removed (%)

79.0

Number of peaks

11332 (qval < 1E-05)