title: CUTandTAG_RPA_NT
organism: Homo sapiens
description: Protocols: CUTandTAG: Cells were harvested using Trypsin and collected by centrifugation at 600g for 5 minutes at room temperature. DIvA (AsiSI-ER-U2OS) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (Invitrogen) and 1µg/mL puromycin at 37°C under a humidified atmosphere with 5% CO2. AsiSI-dependent DSBs were induced by the addition of 300 nM 4-OHT (Sigma, H7904) into the medium for 4 h or 24 h. Inhibitors used were added to the medium 45 minutes prior to 4-OHT treatment. 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 pellets were resuspended in 5 mL of ice-cold NE1 buffer (200 mM Hepes pH 7.9, 10 mM KCl, 20% Glycerol, 0.5mM Spermidine, 0.1% Triton X- 100) with 1X Complete protease (Roche) and phosphatase inhibitors (P5726, Sigma-Aldrich). After 10 minutes of incubation on ice, nuclei were collected by centrifugation at 1000g, 4°C for 5 minutes and resuspended in PBS containing 0.5 mM Spermidine and protease and phosphatase inhibitors. Nuclei were crosslinked by adding formaldehyde (0.2% final concentration) for 2 minutes at RT. Formaldehyde was quenched by adding glycine (125 mM final) and nuclei were recovered by centrifugation at 1000g, 4°C for 10 minutes. Pellets were resuspended in Antibody buffer (20mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA) with protease and phosphatase inhibitors at around 2.106 nuclei/mL. About 100000 nuclei were bound to 3.5 µL of activated concanavalin A (ConA) magnetic beads (Epicypher) in antibody buffer for 15 minutes at room temperature on a rotating wheel. Beads containing nuclei were washed once with Antibody buffer, resuspended in 200 µL of Antibody buffer, transferred to 8-tubes 0.5 mL PCR strips and primary antibodies (typically 1 to 2 µg) were added. Samples were incubated overnight on a rotating wheel at 4°. The following day, 1 µg of the appropriate secondary antibody was added to the samples and incubated 1h at room temperature on a rotating wheel. Nuclei were washed in Wash Buffer 150 (20mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM Spermidine) with protease and phosphatase inhibitors and resuspended in 200 µL of Wash 300 (Wash Buffer 150 with 300 mM NaCl final). 2 µL of pAG-Tn5 (Epicypher) was added, and samples were placed for 1 h on a rotating wheel at room temperature. After washing with the Wash 300 solution, nuclei were resuspended in 50 µL of Tagmentation buffer (Wash 300 with 10 mM MgCl2). Samples were then incubated at 37°C for 1h in a Thermocycler. Following incubation, the liquid was removed, and 5 µL of SDS Release buffer (10mM TAPS pH 8.5, 0.1% SDS) was dropped on the nuclei/beads mixture. Samples were incubated for 1h at 58°C and 15 µL of 10mM TAPS pH 8.5 with 0.67% Triton-X100 was added to the mixture to quench the SDS. CutandTag: 2.5 µL of 10 µM barcoded i7 and i5 primers were added together with 25 µL of NEBNext HiFi 2x PCR Master mix (New England Biolabs). Samples were subjected to 10 cycles of PCR preamplification using the follwing conditions (58°C for 5 minutes, 72°C for 5 minutes, 98°C for 5 minutes then 10 cycles of 98°C for 15 s, 63°C for 10 s and 72°C for 1 minute). To estimate the number of cycles required for PCR amplification 3 µL of the preamplification reaction was subjected to 30 cycles of real time PCR using the following cycling conditions (98°C for 5 minutes then 10 cycles of 98°C for 15 s, 63°C for 10 s). The number of required cycles were dtermined empirically. Following subsequent PCR amplification, samples were purified using SPRISelect beads (Beckman Coulter). Large fragments were first removed with a 0.4X samples to beads ratio and the CUT&Tag library was recovered with a 1.3X sample to beads ratio. Libraries were quantified with Quantus (Promega) and run on a High Sensitivity DNA chip on the Bioanalyzer 2100 instrument (Agilent Technologies).
attributes.tag: ENA first public
attributes.value: 2026-04-18
attributes.tag: INSDC center name
attributes.value: MCD-CBI, University of Toulouse, Toulouse, France
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-15637:CUTandTAG_RPA_NT
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: U2OS
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: osteosarcoma
attributes.tag: genotype
attributes.value: AsiS1-ER
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: bone
attributes.tag: protocol
attributes.value: CUTandTAG
attributes.tag: sample name
attributes.value: E-MTAB-15637:CUTandTAG_RPA_NT
attributes.tag: scientific_name
attributes.value: Homo sapiens