Antigen

Antigen Class

TFs and others

Antigen

CHD8

Cell type

Cell type Class

Neural

Cell type

Neural Stem Cells

MeSH Description

Self-renewing cells that generate the main phenotypes of the nervous system in both the embryo and adult. Neural stem cells are precursors to both NEURONS and NEUROGLIA.

Sample

source_name

human Neural Stem Cells

cell type

human Neural Stem Cells (hNSCs)

chip antibody

CHD8 N terminus

chip antibody vendor

Abcam

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

RNA-seq: Total RNA was extracted using the miRNeasy Micro Kit (217084, Qiagen), as described in the manufacturer’s instructions. The step of DNase I digestion was included to avoid potential DNA contamination. Standard Illumina Multiplexed Paired-End Library Prep performed by the Yale Center for Genome Analysis (YCGA). ChIP-seq: 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. 100 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 10 mg of CHD8 antibody. Samples were incubated overnight at 4°C with rotation. Beads were precipitated on magnet stand and washed five 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, 5mM sodium butyrate) 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).

Sequencing Platform

instrument_model

Illumina HiSeq 2500

hg38

Number of total reads

167424079

Reads aligned (%)

92.5

Duplicates removed (%)

14.2

Number of peaks

39410 (qval < 1E-05)

hg19

Number of total reads

167424079

Reads aligned (%)

91.8

Duplicates removed (%)

15.3

Number of peaks

38667 (qval < 1E-05)