GSM8390869: H3K27me3_TMD8_IRF4_1; Homo sapiens; OTHER
For each sample, nuclei from three technical triplicates replicates were extracted by incubating cells on ice for 10 min in Nuclei Extraction buffer (NE: 20 mM HEPES–KOH, pH 7.9; 10 mM KCl; 0.1% Triton X-100; 20% Glycerol; 0.5mM spermidine; 1x complete protease inhibitor [Roche # 11836170001]), collecting by centrifugation (600 g, 3 min, 4ºC), discarding the supernatant, and resuspending at [100 µl / 500K nuclei] sample in NE buffer. For each target 500K nuclei were immobilized onto Concanavalin-A beads (EpiCypher #21-1401) and incubated overnight (4ºC with gentle rocking) with 0.5 µg of antibody (IgG, H3K4me3, H3K4me1, H3K27me3, and H3K27Ac. CUT&TAG enriched DNA was purified using the Monarch DNA Cleanup kit (New England Biolabs #T1030S), and 10 ng used to prepare sequencing libraries with the Ultra II DNA Library Prep kit (New England Biolabs #E7645S). Libraries were sequenced to 150 base pairs on an Illumina Novaseq using pair-end mode by Novogene (Sacramento, CA).