GSM5076632: Primary lens cells - HIF1a_rep1; Gallus gallus; OTHER
Cleavage Under Targets & Release Using Nuclease (CUT&RUN) was performed on unfixed permeabilized primary lens cells incubated with a HIF1α-specific  ChIP grade antibody (Abcam, ab2185) and an IgG negative control antibody (Invitrogen, prod no: 10500c) to elucidate genome-wide HIF1α-DNA binding complexes. Briefly, primary lens cells were harvested, counted, and pooled into biological duplicates of 500,000 cells each and resuspended in 1.5 ml Wash Buffer without Digitonin (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM Spermidine, Protease Inhibitor). After three rounds of washing and centrifuging for 3 minutes at 600 g, the resuspended cells were washed in 1 ml wash buffer and ConA-coated magnetic beads (Bangs Labroatories, prod no: BP531, 10μl per sample) were added followed by gentle rotation at room temperature for 10 minutes. Each sample was divided into two equal volumes and the supernatants removed using a magnet stand. 150 μl of antibody buffer with 0.1% final concentration digitonin was added to each aliquot with gentle agitation to permeabilize the cells. For each biological sample, one aliquot of ConA-bound cells was incubated with ChIP-grade HIF1α antibody and the other aliquot was incubated with IgG antibody at a 1:100 dilution and rotated at 4 °C overnight. Antibody-bound ConA-bound cells were washed twice with 1 ml wash buffer with 0.1% digitonin. The supernatant was removed and 150 μl of Protein A-MNase (700ng/mL final concentration, Addgene #123461) in wash buffer with digitonin was added to the pellet with gentle agitation and the mix rotated at 4 °C for 1 hour. Cells were washed twice with 1 ml of digitonin wash buffer and once with 1ml low salt rinse buffer (20mM HEPES pH 7.5, 0.5mM Spermidine, 0.05% Digitonin). The supernatant was removed and 200 µl of ice-cold incubation buffer (3.5mM HEPES pH 7.5, 10mM CaCl2, 0.05% Digitonin) added with gentle agitation. The resulting samples were incubated on ice for 5 minutes before removing the supernatant using the magnet stand followed by the addition of 200 µl STOP buffer (170mM NaCl, 20mM EGTA, 0.05% Digitonin, 50ug/mL RNAse A, 25μg/mL Glycogen) with gentle agitation. The samples were incubated for 30 minutes at 37 °C to release CUT&RUN fragments. Following incubation, the supernatant containing CUT&RUN fragments was transferred to a fresh microcentrifuge tube. CUT&RUN fragments were treated with 2 µl 10% SDS and 2.5 µl Proteinase K (20 mg/ml) for 1 hour at 50 °C. DNA was isolated by standard Phenol-Chloroform extraction using Qiagen MaXtract tubes, and the DNA pellet resuspended in 30 µl 1 mM Tris-HCl pH 8 0.1 mM EDTA Library prep was carried out using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB E7645) following the manufacturer's instructions