Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
NOTCH1

Cell type

Cell type Class
Cardiovascular
Cell type
Large pulmonary artery endothelial cells
NA
NA

Attributes by original data submitter

Sample

source_name
Large pulmonary artery endothelial cells
tissue
Large pulmonary artery endothelial cells
passages
7
chip antibody
Notch1 (Cell Signaling Technology, 3608S, lot5)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were trypsinized 48 h after co-culture, and PAECs were cross-linked with 1% formaldehyde (EMD Millipore), for 10 minutes at room temperature. To neutralize formaldehyde, 2M glycine (ThermoFisher Scientific) was added and incubated for 5 minutes at room temperature. The cells were washed with ice cold PBS twice, snap-frozen and stored at -80 °C. For ChIP-DNA preparation, cells were thawed by adding PBS and incubated at 4 °C by rotating. To extract cell nuclei, cells were treated with hypotonic buffer for 10 minutes on ice and then were homogenized using glass homogenizer. Nuclear pellets were resuspended in RIPA buffer (Millipore) and incubate for 30 minutes on ice. Chromatin was sheared with SFX250 Sonifier (Branson, Dunbury, CT) and was immunoprecipitated with Notch1 antibody, H3K27ac antibody (Cell Signaling Technology) and p300 antibody (Santa Cruz Biotechnology) at 4°C overnight on a nutator. To save input sample, 100 ul of sheared nuclear lysate was removed and store overnight at 4 °C. The second day, protein A/G agarose beads (Millipore) were added to the chromatin-antibody complex and incubate for 1 hr at 4°C on a nutator and then the beads were washed 3 times with RIPA buffer and 1 time with PBS. DNA-protein complex bound to the beads were eluted with SDS buffer (Santa Cruz Biotechnology) and incubated at 65 °C for 10 minutes. Supernatant containing ChIP-DNA and input sample were reverse cross-linked by incubating overnight at 65 °C. The third day, ChIP-DNA was treated with RNase A (Qiagen) and proteinase K (ThermoFisher Scientific) and then was purified. The ChIP-DNA samples were end repaired and A-tailed. The illumina TruSeq adapters (illumina, San Diego, CA) were ligated and size-selected from the gel before doing PCR amplification. PCR products were purified and size selected in the gel again.

Sequencing Platform

instrument_model
Illumina HiSeq 4000

hg19

Number of total reads
60064869
Reads aligned (%)
115.0
Duplicates removed (%)
40.5
Number of peaks
694 (qval < 1E-05)

hg38

Number of total reads
60064869
Reads aligned (%)
117.7
Duplicates removed (%)
39.8
Number of peaks
2140 (qval < 1E-05)

Base call quality data from DBCLS SRA