GSM8214548: mESC_H2AK119ub; Homo sapiens; OTHER
CUT&Tag in SU-DIPG-IV cells: Nuclei were prepared from SU-DIPG-IV cells, and up to 5x105 cells were used per individual CUT&Tag reaction using CUT&Tag V3 (https://www.protocols.io/view/cut-amp-tag-direct-for-whole-cells-with-cutac-x54v9mkmzg3e/v3). pAG-Tn5 was purchased from Epicypher (Durham, North Carolina), and H3K27me3 antibody was purchased from Cell Signalling Technologies (#9733). Rabbit Isotype IgG control (ab172730) was purchased from Abcam. Antibodies were used at 1:100 dilution. Sequencing was performed on an Illumina NextSeq 500 using a P1/100 cycle kit with 50bp paired-end reads, targeting 10 million reads per sample. CUT&Tag in HEK293: 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. 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. Library were constructed by tagmentation followed by PCR as described in the extract protocol.