GSM8214536: Async H3K27me3 CUT&RUN Rep1; Mus musculus; OTHER
Genome-wide H3K27me3 domain changes were determined using the CUT&RUN protocol adapted from Hainer & Fazzio. Cells were harvested via 0.25% trypsin-EDTA solution and counted using 0.4% trypan blue stain and Countess automated cell counter (Invitrogen). A single-cell suspension was then prepared to achieve 0.5 x 10^6 cells in 1 mL cold nuclear extraction buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.5 mM spermidine, 0.1% Triton x-100, & 20% glycerol). Isolated nuclei were pelleted via centrifugation at 2000 rpm and 4°C for 5 minutes, then resuspended in 600 µL cold nuclear extraction buffer. Biomag Plus Concanavalin A beads (Bangs Laboratories) were activated via two washes with 1 mL cold binding buffer (20 mM HEPES-KOH pH 7.5, 10 mM KCl, 1 mM CaCl2, & 1 mM MnCl2) using a magnetic rack, with 150 µL bead slurry used for each sample of 0.5 x 106 cells. After the final wash, beads were resuspended in 300 µL cold binding buffer. While gently vortexing, nuclei were added to activated beads and then incubated on a rotating platform at 4°C for 10 minutes to facilitate nuclei binding. Nuclei:bead samples were resuspended in 1 mL blocking buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.1% BSA, 2 mM EDTA, & protease inhibitors) and incubated at room temperature for 5 minutes. After blocking, samples were washed with 1 mL wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.1% BSA, & protease inhibitors) then resuspended in 250 µL wash buffer. While gently vortexing, 5 µg of anti-H3K27me3 (#9733S, Cell Signaling Technology) in 250 µL wash buffer was added to each sample. Antibody binding was facilitated by incubating samples on a rotating platform at 4°C overnight. Samples were washed twice with 1 mL cold wash buffer and then resuspended in 250 µL cold wash buffer. While gently vortexing, 2.5 µL of CUTANA pAG-MNase (EpiCypher) was added, and samples were incubated at 4°C for 1 hour to facilitate binding. After incubation, samples were washed twice with 1 mL cold wash buffer and resuspended in 50 µL cold wash buffer. While gently vortexing, 3 µL 0.1 M CaCl2 was added to each sample. Samples were briefly flicked to mix and then incubated on ice for 30 minutes after which the pAG-MNase was inactivated by adding 50 µL 2x STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, & 0.05% digitonin) supplemented with RNase A (ThermoFisher Scientific), proteinase K (ThermoFisher Scientific), glycogen (VWR), and spike-in DNA generated from MNase digestion of Drosophila S2 cells. Released fragments were cleaned up by incubating samples at 37°C for 20 minutes, followed by centrifugation at 16000xg and 4°C for 5 minutes. The resulting supernatant was mixed with 3 µL 10% SDS and 2.5 µL proteinase K solution, followed by incubation at 70°C for 10 minutes. To purify MNase-digested DNA fragments, samples underwent phenol-chloroform extraction. The final resulting aqueous phase was transferred and incubated with 2 µg glycogen and 750 µL ethanol at -20°C overnight. DNA was pelleted by centrifugation at 16000xg, air-dried, and then resuspended in 0.1x TE buffer. DNA fragments were checked for quality using the Qubit dsDNA HS Assay kit (Invitrogen) and TapeStation High Sensitivity DNA assay kit (Agilent). Sequencing libraries of DNA fragments isolated following CUT&RUN were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs) and the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs; New England BioLabs). Libraries were checked for quality using the Qubit dsDNA HS Assay kit and TapeStation High Sensitivity DNA assay kit. Sequencing was performed using a NovaSeq 6000 with Illumina 150 bp paired-end reads at the Genomics Shared Resource, University of Colorado Cancer Center.