title: GSM9325008: Y-1_fixed_nuclei_histone_DMOG_1; Mus musculus; OTHER
library_name: GSM9325008
library_construction_protocol: Cells were treated with 1 mM DMOG-containing or control medium for 24 hours under standard growth culture conditions and CUT&Tag libraries were prepared using HIF-1α antibodies. Briefly, Y-1 cells were cultured for 24 hours in the presence of 1 mM DMOG or under control conditions. The cells were washed with 1x PBS and treated with trypsin-EDTA (Thermo Fisher Scientific, #15400054) for 4 minutes at 37°C in a cell culture incubator. Trypsin was quenched with cell culture medium and detached cells were transferred to a 15 ml tube and pelleted for 5 minutes at 491 g. The cells were then resuspended in fresh cell culture medium and washed with 1x PBS. Cell pellet was resuspended in ice-cold NE1 buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 0.5 mM spermidine) supplemented with Roche Complete Protease Inhibitor EDTA-Free tablets (Merck, #11836170001) at ½ volume of the starting culture and incubated for 10 minutes on ice. The cell nuclei were pelleted by centrifugation (4 minutes, 1300 g, 4°C) and resuspended in ice-cold 1x PBS. Nuclei were crosslinked with 0.1% formaldehyde (16% (w/v) formaldehyde; Thermo Fisher Scientific, #28906) for 2 minutes at room temperature and the reaction was quenched by glycine solution (70 mM final concentration). Fixed nuclei were then washed with 0.5 ml of nuclei wash buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine) supplemented with Roche Complete Protease Inhibitor EDTA-Free tablets and counted. The nuclear pellet was then resuspended in nuclei wash buffer at a concentration of 1 million nuclei per ml, aliquoted and stored at -80°C until use. The frozen nuclei were thawed at room temperature for 30 minutes and bound to ConA-coated magnetic beads (BioMag Plus Concanavalin A; Bangs Laboratories, #BP531), which were pre-equilibrated with binding buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) in 0.5 ml tubes (3.5 µl beads per 50,000 nuclei) for 10 minutes rotating at room temperature. The supernatant was removed and anti-HIF-1α primary antibody solution (Cayman Chemicals, #10006421) in antibody buffer (CUT&Tag wash buffer with 0.1% BSA) was added at 1:50 dilution. The beads were rotated with the primary Ab solution for 2 hours at RT followed by overnight incubation at 4°C. Next, the supernatant was removed and guinea pig α-rabbit secondary antibody (Antibodies online, #ABIN101961) solution, 1:100 in CUT&Tag wash buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, Roche Complete Protease Inhibitor EDTA-Free tablets; Merck, #11836170001) was added to the beads. The beads were rotated for 1 hour at RT and the supernatant was removed. The beads were then washed with 0.5 ml of CUT&Tag wash buffer and the pA-Tn5 adapter complex (Epicypher, #15-1117) in 300 wash buffer (20 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 0.5 mM spermidine, Roche Complete Protease Inhibitor EDTA-Free tablets; Merck, #11836170001) was added at 1:20. The complex was rotated with the beads for 2 hours at RT and the supernatant was removed. The beads were washed with 0.5 ml of 300 wash buffer and resuspended in CUT&Tag tagmentation buffer (300 wash buffer with 10 mM MgCl2). The samples were incubated for 1 hour at 37°C and cooled down to 8°C in a PCR cycler. The supernatant was removed and beads were washed with TAPS wash buffer (10 mM TAPS pH 8.5, 0.2 mM EDTA). The Tn5 complex was released with 5 µl SDS release solution (10 mM TAPS pH 8.5, 0.1% SDS) at 58°C for 1 h in a PCR cycler. Subsequently, 15 µl Triton neutralisation solution (0.67% Triton X-100) was added per sample. PCR reactions were run on the CUT&Tag samples (gap filling: 5 minutes, 58°C; 5 minutes, 72°C; initial denaturation: 30 s, 98°C; 11 cycles of denaturation 10 s, 98°C; and annealing and elongation 10s, 60°C; final elongation: 1 minute, 72°C; hold 8°C) with barcoded primers using the NEBNext High Fidelity 2x PCR mix (New England Biolabs, #M0541L). DNA was cleaned up with SPRI beads (AMPure XP Bead-Based Reagent; Beckman Coulter, #A63880) for 10 minutes at RT, washed twice with 80% ethanol and eluted with 10 mM Tris-HCl pH 8 for 1 hour at RT. The libraries were subjected to size selection to enrich for fractions between 170 and 700 bp (Fragment Analyzer, Advanced Analytical) and sequenced (Illumina next generation sequencing) at the DRESDEN-concept Genome Center c/o Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden.
identifiers.namespace: GEO
identifiers.id: GSM9325008_r1