GSM4307716: A549, hypoxia, replicate 1, HIF1A ChIP; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
HIF1A
Cell type
Cell type Class
Lung
Cell type
A549
Primary Tissue
Lung
Tissue Diagnosis
Carcinoma
Attributes by original data submitter
Sample
source_name
A549 cells
cell line
A549
cell type
lung adenocarcinoma
treatment
Hypoxia (1% O2 24hrs)
library type
unstranded
chip antibody
Novus Biologicals, NB100-134
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Chromatin immunoprecipitation. Sub-confluent cultures of each cell line (HCT116, RKO, A549, and H460) were placed in normoxic or hypoxic conditions for 24 hours. After treatment, media was removed and replaced with 1% formaldehyde in PBS (equilibrated by bubbling for 20 min with a mix of 1% O2, 5% CO2, and 94% N2 for hypoxia samples). To avoid rapid degradation of HIF1A, a crosslinking time of 5 min was used for all samples and terminated by addition of glycine to 0.125 M final concentration. Following 5 min of formaldehyde quenching, plates were placed on ice and washed three times with ice-cold PBS. Subsequently, cells were lysed in RIPA buffer and sonicated to generate DNA fragments of ~200-300 bp (Qsonica Q800R, 70% amplitude, 30 sec on/30 sec off cycle, 20 cycles for H460 lysates, 25 cycles for HCT116 and RKO lysates, and 30 cycles for A549 lysates). Samples were centrifuged at 20000 g for 20 min at 4°C and protein concentration in collected supernatants was measured with a BCA Protein Assay Kit. At this step, input samples (50 µg of total protein per cell line) were set aside. For each sample, four 1 ml aliquots each containing 1 mg of total protein were used for immunoprecipitation. Samples were pre-cleared with 15 µl of RIPA-washed Dynabeads Protein G (Invitrogen, Thermo Fisher Scientific) by rocking for 1 hour at 4°C. The supernatant was then collected and incubated with 30 µl of Dynabeads and 5 µl of anti-HIF1A antibody (Novus Biologicals, NB100-134) incubated overnight on a rocker at 4°C. Next, beads were washed twice with RIPA buffer, twice with IP wash buffer (500 mM LiCl, 100 mM Tris pH 8.5, 1% v/v NP-40, 1% w/v sodium deoxycholate), and twice with RIPA (2 min on rocker at 4°C for each washing step). Immunocomplexes were eluted by resuspending beads in 100 µl TE buffer and 200 µl of elution buffer (70 mM Tris pH 8, 1 mM EDTA and 1.5% w/v SDS) and incubating for 10 min at 65°C. Both eluted immunocomplexes and input samples were combined with NaCl solution to a final concentration of 200 mM and incubated overnight at 65°C to reverse formaldehyde crosslinks, followed by treatment with 20 µg proteinase K. DNA fragments recovered using phenol/chloroform extraction followed by ethanol precipitation and re-dissolved in TE buffer. Library preparation. DNA fragments were size-selected (80-600 bp) by agarose gel electrophoresis (2% gel, BluePippin) and used for barcoded library preparation with the NEBNext Ultra II DNA kit, according to the manufacturer's instructions (New England Biolabs). Final libraries were size-selected (200-600 bp, BluePippin) and analyzed on Bioanalyzer High Sensitivity DNA chips (Agilent) to confirm fragments ranging from 200 to 400 bp. Single-end 150 bp sequencing of pooled barcoded libraries was carried out on the Illumina HiSeq 4000 platform by the Genomics Core facility at the University of Colorado Anschutz.