Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
TFEB

Cell type

Cell type Class
Cardiovascular
Cell type
HUVEC
Primary Tissue
Umbilical Cord
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
HUVEC
strain
endothelial cells from umbilical cords newborn healthy humans
antibody
TFEB (#4240, CST)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For ChIP-seq, approximately 2 x 10E7 cells were cross-linked by addition of formaldehyde to 1% for 10 min at RT, quenched with 0.125 M glycine for 5 min at RT, and then washed twice in cold PBS. The cells were resuspended in Lysis Buffer 1 (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100 and protease inhibitor) to disrupt the cell membrane and in Lysis Buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and protease inhibitor) to isolate nuclei. The isolated nuclei were then resuspended in SDS ChIP Buffer (20 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS and protease inhibitors). Extracts were sonicated using the Bioruptor H Twin (Diagenode) for 2 runs of 10 cycles [30 sec ‘‘ON’’, 30 sec ‘‘OFF’’] at high power setting. Cell lysate was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was diluted with ChIP Dilution Buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton) before immunoprecipitation step. Magnetic beads (Dynabeads® Rat Anti-Mouse IgM for anti-PolII-phospho-S5, Dynabeads®Protein G for all the other ChIPs, Life Technologies) were saturated with PBS/1% BSA and the samples were incubated with 2 micrograms of antibody overnight at 4°C on a rotator. Next day samples were incubated with saturated beads for two hours at 4°C on a rotator. Successively immunoprecipitated complexes were washed five times with RIPA buffer (50 mM Hepes-KOH pH7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0,7% Na-Deoxycholate) at 4°C for 5 minutes each on a rotator. Elution Buffer was added and incubated at 65°C for 15 minutes. The decrosslinking was performed at 65°C overnight. Decrosslinked DNA was purified using QIAQuick PCR Purification Kit (QIAGEN) according to the manufacture’s instruction. For ChIP-seq, approximately 10 ng of purified ChIP DNA were end-repaired, dA-tailed, and adapter-ligated using the NEBNext ChIP-Seq Library Prep Master Mix Set (NEB), following manufacturer instructions.

Sequencing Platform

instrument_model
NextSeq 500

hg19

Number of total reads
22825340
Reads aligned (%)
46.7
Duplicates removed (%)
50.4
Number of peaks
4865 (qval < 1E-05)

hg38

Number of total reads
22825340
Reads aligned (%)
48.3
Duplicates removed (%)
49.1
Number of peaks
4789 (qval < 1E-05)

Base call quality data from DBCLS SRA