DRIP_siCTRL
Protocols: DRIP-Seq: DIvA cells were trypsinized, pelleted at low speed and washed with DPBS (Life technology). DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (InVitrogenInvitrogen) with 1 µg/mL puromycin (DIvA cells) at 37°C under a humidified atmosphere with 5% CO2. siRNA transfections were performed using 4D-Nucleofector from Lonza either with the SE-small kit for 1 million cells or SE-large kit for 5-20 million of cells, according to the manufacturer's instruction. Cells were collected 48 h after siRNA transfection.  Cell lysis was performed by incubation in cell lysis buffer (5 mM Pipes pH 8, 85 mM KCl, 0.5% IGEPAL® CA-630) followed by Dounce homogenization. Nuclei were harvested by centrifugation and incubated in nuclear lysis buffer: (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS) and sonicated 10 times for 10 s at a power setting of 5 and 50% duty cycle (Branson Sonifier 250). Samples were diluted 10 times in ChIP dilution buffer (16.7 mM Tris pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X‐100, 0.01% SDS) and precleared using a mixture of protein A-agarose (Pierce) and protein G-sepharose (Sigma) previously blocked with BSA (Sigma). Precleared samples (10 to 200 μg) were incubated overnight at 4°C with indicated antibodies. Immune complexes were recovered by incubation with blocked protein A/protein G beads for 2 h at 4°C on a rotating wheel. Beads were washed once in dialysis buffer (50 mM Tris pH 8, 2 mM EDTA, 0.2% Sarkosyl), five times in wash buffer (100 mM Tris pH 8.8, 500 mM LiCl, 1% IGEPAL® CA-630, 1% NaDoc) and twice with TE (10mM Tris-HCl, pH 8, 1mM EDTA). Beads were resuspended in TE supplemented with 50µg/ml DNase-free RNase A (Abcam) and incubated for 30 minutes at 37°C. SDS (final concentration 0.5%) was added and crosslink was reversed by overnight incubation at 70°C. Following a 2 hours treatment with 200 µg/ml proteinase K (Roche) at 45°C, DNA was purified by phenol/chloroform extraction and recovered by ethanol precipitation in presence of 5 µg glycogen (Invitrogen). DNA pellet was resuspended in water. Prior to next-generation sequencing library preparation, samples from multiples ChIP experiments were pooled and sonicated for 5 cycles (30 seconds on, 30 seconds off, high setting) on a Bioruptor (Diagenode) then concentrated with a vacuum concentrator (Eppendorf). For DRIP: Total nucleic acids were extracted with 0,5% SDS /Proteinase K (Thermo Fisher Scientific, Waltham, MA) treatment at 37°C overnight and recovered by phenol-chloroform extraction and ethanol precipitation. DNA was digested by a restriction enzyme cocktail (20 units each of EcoRI, HindIII, BsrGI, XbaI) (New England Biolabs) in 1× NEBuffer 2, with or without RNase H treatment, overnight at 37°C. Fragmented DNA was cleaned by phenol-chloroform extraction and ethanol precipitation followed by two washes with 70% ethanol. Air-dried pellets were resuspended in 10 mM Tris-HCl pH 7.5, 1 mM EDTA (TE). 4 µg of digests was diluted in 450 µL of TE, and 10 µL was reserved as input for qPCR. 50 µL of 10× IP buffer was added (final buffer concentration of 10 mM sodium phosphate, 140 mM sodium chloride, 0.05% Triton X-100) and 10 µL of S9.6 antibody (1 mg/ml, kind gift from F. Chedin, UC Davis). Samples were incubated with the antibody at 4°C for 2 hours on a wheel. 50 µL of Protein A/G Agarose (Pierce), previously washed twice with 700 µL of 1× IP buffer for 5 minutes at room temperature, were added and samples were incubated for 2h at 4°C on a wheel. Each DRIP was then washed three times with 700 µL 1× IP buffer for 10 minutes at room temperature. After the final wash, beads were resuspended in 250 µL of 1× IP buffer and incubated with 60 units of Proteinase K for 45 minutes at 55°C. Digested DRIP samples were then cleaned with phenol-chloroform extraction and ethanol precipitation. Air-dried DRIP pellets were resuspended in 45 µL of 10 mM Tris-HCl pH 8. Library preparation was perfomed at EMBL Genomics core facilities (Heidelberg, Germany) using NEBNext ChIP-Seq Library Prep kit (New England Biolabs).
ENA first public
2026-07-31
INSDC center name
CBI, MCD, University of Toulouse, FRANCE
INSDC status
public
Submitter Id
E-MTAB-15750:DRIP_siCTRL
broker name
ArrayExpress
cell line
U2OS
cell type
epithelial cell
collection date
not collected
common name
human
disease
osteosarcoma
genotype
AsIS1-ER
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
bone
sample name
E-MTAB-15750:DRIP_siCTRL
scientific_name
Homo sapiens