ChIP Sequencing of Crispr corrected Bloom Syndrome patient cell line using antibody against BLM
ChIP Sequencing of Crispr corrected Bloom Syndrome patient cell line using antibody against BLM
10 million cells for each ChIP were cross linked using formaldehyde at 1% final concentration, followed by its neutralization using 1.25M Glycine solution. The cross-linked chromatin was thoroughly washed with ice cold PBS and lysed using Lysis Buffer (1% SDS, 10mM EDTA, 50mM Tris pH8.1) followed by sonication using DiagenodeBioruptor Pico to obtain average length between 200-1000bp. 200µg of chromatin was incubated overnight with 3µg of either BLM or RAD54 antibodies, followed by immunoprecipitated using pre-blocked Protein A/G beads (blocked with with 100 mg/mL BSA and 500 mg/mL salmon sperm DNA) for 4 hours at 4⁰C in a rotating platform. The beads were then washed with low salt buffer (0.01% SDS, 2mM EDTA, 20mM Tris-Cl pH 8.1, 1% Triton X-100 and 150mM NaCl), high salt buffer (same as low salt except 500mM NaCl instead of 150mM), lithium chloride buffer (0.25M LiCl, 2% IGEPAL, 1% sodium deoxycholic acid, 1mM EDTA and 10mM Tris-Cl pH 8.1) once followed by two washed with Tris-EDTA (10mM Tris pH 8.0 and 1mM EDTA pH 8.0). The eluted chromatin was reverse cross-linked using Proteinase K digestion (10μg/ml) at 55⁰C for 45 minutes, followed by incubation at 80⁰C. Subclone 9.6 cells were grown in DMEM+10% FBS+antibiotics. Eluted DNA was recovered through Phenol: Chloroform: Iso-amylalcohol and precipitated using isopropanol and 100mg of Glycogen and dissolved in nuclease free water. The concentrations of input samples and ChIP DNA will be determined by Qubit using dsDNA HS kit. Library prep kit used for ChIP - *NEBNext® Ultra™ II DNA Library Prep with Sample Purification Beads (Cat. No-E7103L).