title: CutnRun directed against O-GlcNac in the human DLD-1 cell line with doxycycline treatment to induce RNA Polymerase II degradation
design: CutnRun directed against O-GlcNac in the human DLD-1 cell line with doxycycline treatment to induce RNA Polymerase II degradation
library_construction_protocol: For each condition, 1x106 cells were collected and centrifuged at 600 g for 3 min at 4°C. Human colon adenocarcinoma DLD-1 cells expressing OsTIR and with a cassette encoding mini-AID (mAID) and fluorescent protein mClover (mAID+mClover) at the initiation site of the endogenous Rpb1 gene locus (POLR2A) (Nagashima 2019) were grown in RPMI 1640 medium (Thermo Fisher Scientific #A1049101) supplemented with 10% FBS (Gibco). Degradation of Pol II was induced with Doxocycline. Both wild-type and treated data are provided in this dataset. Cleavage under targets and release using nuclease (CUT&RUN) was performed as previously described on live nuclei (Skene 2017). Nuclei were extracted by resuspending the cell pellet in 1 mL of Nuclear Extraction Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.5 mM Spermidine, 0.1% Triton X-100, 20% Glycerol, 1X cOmplete Mini EDTA-Free Protease Inhibitors (Roche)). 25 μL of Concanavalin A magnetic beads (Bangs Laboratories #BP531) previously washed twice in Binding Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2) were gently added to each nuclei sample and incubated for 10 min at RT under gentle rotation. The bead-bound nuclei were isolated on a magnetic stand and beads were blocked with 1 mL of Blocking Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, 2 mM EDTA, 1X cOmplete Mini EDTA-Free Protease Inhibitors (Roche)) for 5 min at RT. The bead-bound nuclei were isolated on the magnetic stand, washed with 1 mL of Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, 1X cOmplete Mini EDTA-Free Protease Inhibitors (Roche)), incubated for 5 min with 1 mL of Wash Buffer supplemented with 0.1% of Triton X-100, subsequently washed twice with Wash Buffer and resuspended in 300 μL of Antibody Buffer with 0.01% TritonX-100 supplemented with 1 μg of antibody. Samples were incubated under gentle rotation overnight at 4°C, washed twice with 1 mL of Wash Buffer and resuspended in 300 μL of Wash Buffer with 0.01% TritonX-100 supplemented with 700 ng/mL of pA/G MNase. The bead-bound nuclei were incubated for 1h at 4°C under gentle rotation and washed twice with Wash Buffer. The samples were resuspended in 50 μL of Wash Buffer and placed in iced water to pre-cool to 0°C. pA/G MNase targeted digestion was initiated by adding 2 μL of CaCl2, samples were mixed by flicking and incubated for 30 min on iced water at 0°C. Digestion reaction was stopped by adding 50 μL of 2X Stop Buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 1% IGEPAL, 1 mM MnCl2). Samples were incubated at 37°C for 15 min to release the CUT&RUN fragments from the insoluble nuclear chromatin and centrifuged at 16,000 g for 5 min at 4°C. Supernatants were collected after magnetic beads separation on magnetic stand and transferred to new tubes. 2 μL of 10% SDS and 2.5 μL of 20 mg/ml of  Protein K were added and samples were incubated at 70°C for 10 min. DNA fragments were purified and size selected using SPRIselect magnetic beads (Beckman Coulter #B23318) following the manufacturer's protocol for single selection to purify fragments higher than 100 bp. DNA fragments were eluted in 30 μL of 0.1 M TE.  Samples concentrations were verified using Qubit III and libraries prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturer's protocol. Libraries were size selected from Nusieve 3:1 Agarose gel to isolate fragments from 250 base pair (bp) to 460 bp and DNA purified using Monarcharch DNA Gel Extraction Kit Protocol (New England Laboratories #T1020). Libraries quality was evaluated using Tape Station DNA HS D1000 Kit on a Tape Station system (Agilent 4150).