title: GSM9491672: X2PSM_CT_rep1; Mus musculus; OTHER
library_construction_protocol: The CUT&RUN of CBX2 in CBX2fl/fl-HaloTag was performed using a CUTANA™ ChIC/CUT&RUN Kit (EpiCypher). Cells were harvested, split to a single-cell suspension, counted, and 0.5 x 10^6 cells were used in each reaction, and reactions were run in duplicate. Cells were washed with PBS, then Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 x EDTA-free protease Inhibitor), and bound to activated ConA beads for 10 minutes. Supernatant was removed from beads, 50 μL Antibody Buffer ((20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 x EDTA-free protease Inhibitor, 0.1% Digitonin, 2 mM EDTA) with 1:50 Anti-HaloTag® (polyclonal) (Promega) or 1:200 Anti-GFP (monoclonal) (Invitrogen) was added, and cells were gently vortexed to thoroughly resuspend beads before overnight incubation at 4°C on a nutator. The following day, the Antibody buffer was removed with the tubes on a magnetic stand and cells were washed twice with pAG-MNase Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 x EDTA-free protease Inhibitor, 0.1% Digitonin). After the washes, 50 μL pAG-MNase Buffer + 2.5 μL pAG-MNase was added to each reaction, tubes were then vortexed to mix and incubated at RT for 10 minutes. Supernatant was removed and each reaction was washed twice with 200 μL pAG-MNase Buffer. Afterwards 2 mM CaCl2 in pAG-MNase Buffer was added to activate pAG-MNase for chromatin digestion, beads were vortexed to resuspend, and tubes were then incubated at 4°C on a nutator for 2 hours. Stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A, 50 μg/mL Glycogen) + E. coli spike-in was added following digestion and tubes were gently vortexed to mixed then placed in a preheated thermocycler set to 37°C. Reactions were then purified and eluted from included columns, then quantified using a Qubit.  Genome-wide H3K27me3 domain changes were profiled using the CUT&Tag protocol adapted from EpiCypher. Cells were harvested via 0.25% trypsin-EDTA solution and counted using 0.4% trypan blue stain and Countess automated cell counter. A single-cell suspension was prepared by washing cells with cold 1x PBS and resuspending 1.0 x 106 cells in 1 mL cold nuclear extraction buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.1% Triton x-100, 20% glycerol, & 0.5 mM spermidine), then incubated on ice for 10 minutes to isolate nuclei. Biomag Plus Concanavalin A beads were activated with two washes of cold bead activation buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2, & 1 mM MnCl22). After activation, beads were added to aliquots of 0.1 x 106 isolated nuclei and incubated at room temperature for 10 minutes. Nuclei:bead samples were resuspended in 50 µL cold Antibody150 buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, 2 mM EDTA, & protease inhibitors) followed by the addition of 0.5 µg of anti-H3K27me3 (#9733S, Cell Signaling Technology). To facilitate primary antibody binding, samples were incubated on nutator at room temperature for 1 hour. Samples were resuspended in 50 µL Digitonin150 buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, & protease inhibitors) followed by the addition of 0.5 µg CUTANA anti-rabbit secondary antibody (#13-1047, EpiCypher). Samples were then incubated on nutator at room temperature for 1 hour to facilitate binding, followed by two washes with 200 µL cold Digitonin300 buffer (20 mM HEPES-KOH pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, & protease inhibitors). The tagmentation reaction was performed by resuspending samples in 50 µL cold Tagmentation buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 10 mM MgCl2, & protease inhibitors) and incubating at 37°C in thermocycler for 1 hour. Samples were washed with 50 µL TAPS buffer (10 mM TAPS pH 8.5 & 0.2 mM EDTA) then resuspended in 5 µL SDS Release buffer (10 mM TAPS pH 8.5 & 0.1% SDS) to quench the tagmentation reaction and release adaptor-tagged DNA fragments into solution. For CUT&RUN, following end repair and adaptor ligation, 1X SPRIselect beads were used to wash prepped DNA and PCR was ran for 15 cycles of denaturation and annealing/extension. The PCR settings were: Initial Denaturation at 98°C for 30 Seconds, Denaturation at 98°C for 10 seconds, Annealing/extension at 65°C for 75 seconds, Final Extension at 65°C for 5 minutes. Following PCR, 1X SPRIselect beads were used to wash amplified library-prepped DNA and each eluted product was ran on a Tapestation (Agilent) for fragment analysis before multiplexing and PE150 sequencing. For CUT&Tag, After incubating samples at 58°C in thermocycler for 1 hour, 15 µL SDS Quench buffer (0.67% Triton x-100), 2 µL i5 universal primer (EpiCypher), 2 µL i7 barcoded primer (EpiCypher), 0.005 ng Escherichia coli spike-in DNA (made in-house), and 25 µL NEBNExt High Fidelity 2x PCR Master Mix were added to each sample for library construction with the following PCR protocol: 5 minutes at 58°C, 5 minutes at 72°C, 45 seconds at 98°C, 14 cycles of 15 seconds at 98°C and 10 seconds at 60°C, with a final extension for 1 minute at 72°C. Libraries were checked for quality using the Qubit dsDNA HS Assay kit and TapeStation High Sensitivity DNA assay kit and then sequencing was performed using Novaseq 6000 (Novogene) to generate 150 bp paired-end reads.