GSM9822503: Sojd_CHR_H3K27me3_Passage_38; Homo sapiens; OTHER
CUT&Tag was carried out using EpiCypher CUTANA™ CUT&Tag Protocol v 1.7. 120,000 cells per CUT&Tag experiment were harvested for nuclei extraction. The cell suspension was centrifuged for 3 minutes at 300 RCF at RT. The cell pellet was resuspended in 100 µl/sample of cold nuclei extraction buffer (20 mM HEPES–KOH, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5 mM Spermidine (Sigma-Aldrich, 05292), 1x Roche cOmpleteTM, Mini, EDTA-free Protease Inhibitor) and incubated on ice for 10 minutes, followed by centrifugation for 3 minutes at 600 RCF and 4°C. The nuclei pellet was resuspended in nuclei extraction buffer to achieve a final concentration of 1.2 million nuclei/ml and frozen at -80°C until library preparation. Upon thawing of the nuclei suspension, 100µl of the suspension (120,000 nuclei) was aliquoted to be used per CUT&Tag reaction. Concavalin A (ConA) beads (EpiCypher, 21-1401) were transferred to a 1.5ml Eppendorf tube on a magnetic stand and washed twice with cold bead activation buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). Beads and buffer were added at 11µl and 100µl per sample, respectively, and after washing, the beads were resuspended in a final volume of 11μl of buffer per sample. 10µl of ConA bead slurry was aliquoted into each sample tube of an 8-strip tube containing 100µl nuclei per tube for batch processing. The ConA bead and nuclei mixture was vortexed gently to mix and incubated at RT for 10 minutes. The tubes were placed on a magnetic stand and the supernatant removed. Each nuclei sample was resuspended in 50µl of cold Antibody150 buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmpleteTM, Mini, EDTA-free Protease Inhibitor, 0.01% digitonin (Sigma-Aldrich, D141-100MG), 2mM EDTA) containing primary antibody (anti-H3K27me3 (Active Motif; 39157; dilution 1:50), anti-H3K4me3 (Active Motif; 39160; dilution 1:50), anti-IgG negative control antibody (EpiCypher; 13-0042; dilution 1:50). Samples were then gently mixed and incubated overnight at 4°C on an orbital shaker. The next day, the tubes were placed on a magnet and the supernatant discarded. Each sample was resuspended in 50µl Digitonin150 buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmpleteTM, Mini, EDTA-free Protease Inhibitor, 0.01% digitonin) containing 0.5μg of secondary antibody (anti-rabbit Epicypher; 13-0047) for 1 hr at RT on a nutator. After the incubation, the samples were placed on a magnet and washed twice with Digitonin150 buffer. The samples were then resuspended in 50µl Digitonin300 buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmpleteTM, Mini, EDTA-free Protease Inhibitor, 0.01% digitonin), and 1.25μl of CUTANA pAG-TN5 (EpiCypher, 15-1017) was added. Samples were then gently mixed and incubated for 1hr at RT on a nutator. After the incubation, the samples were placed on a magnet and washed twice with Digitonin300 buffer (20mM HEPES, pH 7.5, 300mM NaCl, 0.5mM Spermidine, 1x Roche cOmpleteTM, Mini, EDTA-free Protease Inhibitor, 0.01% digitonin). The samples were then resuspended in 50 µl of Tagmentation buffer (Digitonin 300 Buffer, 10 mM MgCl2) and incubated for 1hr at 37°C in a thermocycler. Then the sample tubes were placed on a magnet and the supernatant discarded, the samples were then resuspended in 50µl TAPS buffer (10 mM TAPS, pH 8.5, 0.2 mM EDTA). The tubes were returned to the magnet and the supernatant removed, followed by addition of 5µl SDS release buffer (0.5M HEPES-NaOH pH 8.5, 10% SDS). The reaction mixture was vortexed on maximum speed for 10s and briefly centrifuged. The samples were then incubated on the thermocycler for 1hr at 58℃. The SDS was neutralised by the addition of 15µl SDS quench buffer (0.67% Triton-X 100), vortexed at maximum speed for 10s, and centrifuged briefly. The libraries were amplified using KAPA HiFi HotStart ReadyMix (Roche Diagnostics Ltd, KK2602). 25ul KAPA HiFi HotStart ReadyMix per CUT&Tag reaction (mastermix only) was incubated at 95℃ for 3 min and cooled to 22℃ in a thermocycler. 1μl of each of barcoded i5 and i7 primers (5μM stock) and 25µl of the previously heated mastermix was added to the DNA. PCR was conducted using the following programme: extension step at 72°C for 5 min, denaturation at 98°C for 45 s, 13-15 cycles (denaturation at 98°C for 10s and short extension at 60°C for 10s), and final extension at 72°C for 1 min. Amplified libraries were cleaned twice using 1X volume of AMPure XP Beads (Beckman Coulter; A63881) and resuspended in 15μl of 0.1X TE buffer. Size distribution and yield quantification of the CUT&Tag libraries were tested using an Agilent 2100 Bioanalyzer high sensitivity DNA chip (Agilent 5067–4626). Libraries were then multiplexed and sequenced (paired-end 150 bp) on a NovaSeq X Plus Series PE150 (Illumina) to a depth of a minimum 10 million reads per replicate.