Understanding how cohesin makes DNA loops in the human genome and its role in Cornelia de Lange syndrome CNIO_Cancer NatureComms
MCF10A cells were grown at high confluence in order to arrest them in G1. Cells in suspension were crosslinked with 1% formaldehyde added to the media for 15 min at RT. After quenching the reaction with 0.125 M Glycine, fixed cells were washed twice with PBS containing 1 μM PMSF and protease inhibitors. For chromatin preparation two different protocols were applied depending on the experiment. For experiments labeled in blue in Supplementary Tablecells/ml.
In all cases, chromatin from 10cells was used per immunoprecipitation reaction with 50 μg of cohesin antibodies or 15 μg of CTCF antibody as described. For calibration, 5% of chromatin from mouse ES cells was added to the human chromatin. For library preparation, at least 5 ng of DNA were processed through subsequent enzymatic treatments with “NEBNext Ultra II FS DNA Library Prep Kit for Illumina” from New England BioLabs .
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