Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Embryo
Cell type
Embryonic brains
NA
NA

Attributes by original data submitter

Sample

source_name
Mouse e17.5 Embryonic Occipital Cortex
strain
C57BL6J
developmental stage
Embryonic Day e17.5
tissue
Embryonic Occipital Cortex
experiment type
ChIP-seq
chip antibody
N/A

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Nuclei were extracted in six pellet volumes of lysis buffer I (50 mM Tris pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x Roche Complete Protease inhibitor, 5 mM sodium butyrate) and incubating on ice for 15 minutes. Tissue was homogenized with a dounce homogenizer and nuclei were harvested by centrifugation at 2000g for 10 minutes at 4°C. Nuclei were resuspended in five pellet volumes of nuclear lysis buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 0.2% SDS, 1x Roche Complete Protease inhibitor, 5 mM sodium butyrate) and incubated on ice for 20 minutes. 300 mL nuclear lysates in 1.5 mL tubes were sonicated using a Misonix S4000 with 431A cup horn (10 second pulses, 10 second rest, 20 minutes total, 2°C maintained by circulating chiller). Following sonication insoluble material was pelleted by centrifugation at 16000g for 10 minutes at 4°C. A small sample of soluble crosslinked chromatin was purified, checked for correct sonication size range and DNA concentration was measured. For H3K27ac, 10-25 ug of chromatin was diluted to 450 mL with dilution buffer (16.7 mM Tris pH 8, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, 1x Roche Complete Protease inhibitor, 5 mM sodium butyrate) and added to 50 uL of Protein G Dynabeads (Invitrogen) prebound with 2 mg of H3K27ac antibody.For H3K4me2, 10-25 ug of chromatin was diluted to 450 mL with dilution buffer (16.7 mM Tris pH 8, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, 1x Roche Complete Protease inhibitor) and added to 50 uL of Protein G Dynabeads (Invitrogen) prebound with 10 mg of H3K4me2 antibody. Samples were incubated overnight at 4°C with rotation. Beads were precipitated on magnet stand and washed eight times with 1 mL of wash buffer (100 mM Tris pH8.0, 500 mM LiCl, 1% NP-40, 1% deoxycholic acid, 1x Roche Complete protease inhibitor) and once with TE. Immunoprecipiated chromatin was eluted from beads with 50 mL of elution buffer (TE + 1% SDS) at 65°C for 10 minutes. Crosslinks were reversed overnight at 65°C. Chromatin was treated with RNAseA and proteinase K then purified with Qiagen PCR cleanup kit. Concentration of eluted chromatin was measured with PicoGreen (Invitrogen). Standard Illumina Multiplexed Paired-End Library Prep

Sequencing Platform

instrument_model
Illumina HiSeq 2500

mm10

Number of total reads
41011916
Reads aligned (%)
97.5
Duplicates removed (%)
14.0
Number of peaks
445 (qval < 1E-05)

mm9

Number of total reads
41011916
Reads aligned (%)
97.3
Duplicates removed (%)
14.0
Number of peaks
468 (qval < 1E-05)

Base call quality data from DBCLS SRA