GSM7663248: A549 Ythdc1 ChIP, rep3; Homo sapiens; ChIP-Seq
30x106 A549 cells were crosslinked with 1% formaldehyde in 1X PBS for 10 minutes in agitation. Crosslinking was quenched with final concentration of 125 mM Glycine (Bio-Rad). Cells were collected and incubated in 7 mL of Cell Lysis Buffer (5 mM Tris [pH 8], 85 mM KCl, 0.5% IGEPAL) for 10 minutes at 4ºC in rotation. Nuclear samples was collected by centrigation for 10 minutes at 3000 xg and 4ºC. Pellet was washed in Cell Lysis Buffer without IGEPAL (5 mM Tris [pH 8], 85 mM KCl) and pelleted again. Pellet was resuspended in RIPA Buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 0.1% SDS, 0.5% Na-Deoxycholate [Na-DOC], 1% Triton X-100) supplemented with 0.1 mM DTT, 1X cOmplete Protease Inhibitor Cocktail (Roche) and 20U/mL RNAsin Ribonuclease Inhibitors (Promega) for 10 minutes in ice. Lysate was dounced and chromatin was sheared by sonication in a Bioruptor device for 45 cycles (30''ON-30''OFF). We cleared nuclear extract by centrifugating 30 minutes at max speed and incubation with 50 µL Protein A/G Dynabeads for 1 hour at 4ºC. Nuclear extract was incubated with primary antibodies (see Table antibodies) overnight at 4ºC in rotation. The following day, 50 µL Protein A/G Dynabeads where washed and mixed to antibody-hybridized nuclear extract for 6 hours at 4ºC. Beads were washed following the next order: 1 wash of Low-Salt Buffer (150 mM NaCl, 20 mM Tris [pH 8], 2 mM EDTA, 0.1% SDS, 1% Triton x-100), 1 wash of High-Buffer (500 mM NaCl, 20 mM Tris [pH 8], 2 mM EDTA, 0.1% SDS, 1% Triton x-100), 1 wash of LiCl Buffer (0.25 M LiCl, 1% IGEPAL, 1% Na-Deoxycholate [Na-DOC], 1 mM EDTA, 10 mM Tris [pH 8]) and 2 washes with TE Buffer (10 mM Tris, 1 mM EDTA [pH 8]). Beads are resuspended in 250 µL of Elution Buffer (25 mM Tris [pH 7.5], 5 mM EDTA, 0.5% SDS) supplemented with 10 µL of 5 M NaCl, 10 µL 1M Tris pH 7, 5 uL 0.5 M EDTA and 20 µg Proteinase K for 4 hours at 65ºC to promote proper decrosslinking of chromatin from immunoprecipitated proteins. DNA was isolated with Phenol:Chloroform:isoamyl (25:24:1) alcohol (Sigma-Aldrich) isolation and quantified by Qubit4 fluorometer (Invitrogen) Custom protocol