Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

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.

Attributes by original data submitter

Sample

source_name
NS5 Neural Stem Cell
cell type
NS5 Neural Stem Cell
chip antibody
Goat IgG (Santa Cruz: sc2028)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For each ChIP 1.5*10^8 NSCs were used. As a control, the same experiment was performed in parallel using goat IgG. NSCs were crosslinked with 2 mM disuccinimidyl glutarate (DSG, Thermo Fisher Scientific) for 45 min at RT, washed three times (PBS + 1mM PMSF) and then crosslinked for 10min with 1% formaldehyde. This reaction was quenched by addition of 1/20 volume of 2.5M glycine for 10min at RT. Cells were washed twice with cold 1x PBS+1mM PMSF, collected by centrifugation, and cell pellets frozen with N2(l). Cells were resuspended in LB1 (50mM Hepes-KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP40, 0.25% Triton-X-100 . After 10 minutes incubation, cells were pelleted by centrifugation and resuspended in LB2 (10 mM Tris-HCl, pH8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA. After 10 minutes incubation, cells were pelleted and resuspended in freshly prepared LB3 (10mM Tris-HCl pH8.0, 100mM NaCl, 1mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine and sonicated on a Soniprep 150(MSE). For quality control of the sonication, it was verified whether the DNA fragment size was between 100 and 1000bp. The DNA concentration was measured in order to take a volume of chromatin solution containing 300µg DNA per ChIP. Per sample, 150µl prot-G Dynabead solution (Life Technologies) was used. Beads were blocked for 1hr at RT with 0.5mg/ml BSA and 0.2mg/ml sonicated salmon sperm DNA in IP buffer (20mM Tris-HCl pH8, 150mM NaCl, 2mM EDTA, 1% Triton X-100) After blocking, the beads were washed 3 times in IP buffer. To lower unspecific binding, the chromatin was incubated with beads without the presence of antibody, rotating for 30 min at 4 C. The beads were discarded and the 'precleared' chromatin was incubated overnight at 4 C with 15µg of antibody. The next day, 150µl of blocked beads was added to this mixture and rotated for 3hrs at 4 C. The supernatant was discarded and the beads were washed subsequently by rotation for 10min for each wash: 2x low salt buffer(20mM Tris-HCl pH8, 150mM NaCl, 2mM EDTA, 1% Triton X-100), 2x high salt buffer (20mM Tris-HCl pH8, 500mM NaCl, 2mM EDTA, 1%Triton X-100, 0.1% SDS), 2x LiCl buffer (10mM Tris-HCl pH8, 250mM LiCl, 1mM EDTA, 0.5% NP-40, 0.5% deoxycholate) and finally twice in TE (10 mM Tris-HCl pH8, 1mM EDTA). Immune complexes were eluted in 210ul elution buffer (1% SDS, 0.1 M NaHCO3) for 1 h at 65 °C while softly shaking. The crosslinks were reversed by incubation overnight at 65C. The following day, the samples were treated with proteinase K for one hour at 45C. DNA was extracted by phenol/chloroform/isoamyl extraction and precipitated with ethanol. The library was prepared according to the illumina protocol. Briefly 10 ng of DNA ChIP material was end-repaired, ligated to adapters, size selected on gel (200 bp +-25 bp range) and PCR amplified using Phusion polymerase with 30 seconds incubation at 98 ᴼC, 18 cycles of 10 second 98 ᴼC , 30 seconds 65 ᴼC, 30 seconds 72ᴼC. Followed with 5 minutes of final extension at 72 ᴼC. Cluster generation was performed using the Illumina HiSeq 2000 platform.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
24786226
Reads aligned (%)
89.0
Duplicates removed (%)
34.4
Number of peaks
579 (qval < 1E-05)

mm9

Number of total reads
24786226
Reads aligned (%)
88.6
Duplicates removed (%)
34.5
Number of peaks
639 (qval < 1E-05)

Base call quality data from DBCLS SRA