Antigen

Antigen Class

TFs and others

Antigen

Npas3

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

NS5 Neural Stem Cell

cell type

NS5 Neural Stem cell

chip antibody

Rabbit Anti-Npas3 (Sigma: HPA002892)

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 rabbit 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, that was next diluted 1 to 5 with ChIP dilution buffer (20mM Tris-HCl pH8, 150mM NaCl, 2mM EDTA, 1% Triton X-100 and 1x CEF protease inhibitor). Equilibration of preblocked protein G beads (Millipore) was performed by washing them 3x with 1 ml of ChIP dilution buffer. To lower unspecific binding, the diluted 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, 60µl of 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 5min for each wash: 1x ChIP dilution buffer, 1x high salt buffer (20mM Tris-HCl pH8, 500mM NaCl, 2mM EDTA, 1%Triton X-100, 0.1% SDS),1x LiCl buffer (10mM Tris-HCl pH8, 250mM LiCl, 1mM EDTA, 0.5% NP-40, 0.5% deoxycholate) and twice in TE (10 mM Tris-HCl pH8, 1mM EDTA). Elution was performed two times by incubation in 250 l of pre-warmed (37C) freshly prepared elution buffer (0.1M NaHCO3, 1% SDS) for 15 minutes at RT. Supernatants were combined and centrifuged to get rid of beads. 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

24575920

Reads aligned (%)

94.7

Duplicates removed (%)

34.2

Number of peaks

16958 (qval < 1E-05)

mm9

Number of total reads

24575920

Reads aligned (%)

94.4

Duplicates removed (%)

34.3

Number of peaks

16922 (qval < 1E-05)