GSM9218832: T47DS_GRHL2_R5020_rep2; Homo sapiens; OTHER
CUT&RUN LoV-U was performed as described in Zambanini et al, with the following modifications. Approximately 500,000 T47DS cells per sample were harvested using 0.25% trypsin, and cells were washed three times in nuclear extraction buffer (20 mM HEPES-KOH (pH 8.2), 10 mM KCl, 0.5 mM spermidine, 0.05% IGEPAL, 20% glycerol, and Roche Complete Protease Inhibitor EDTA-Free) to isolate nuclei. Nucleic pellets were flash-frozen in an isopropyl chamber and stored at –80 °C until further processing. Nuclei were thawed, washed once in nuclear extraction buffer and subsequently bound to equilibrated magnetic ConA agarose beads. Following incubation, nuclei-bead complexes were washed for 5 minutes in wash buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, and Roche Complete Protease Inhibitor EDTA-Free, 0.025% digitonin) supplemented with 2 mM EDTA, and then transferred to PCR tubes. Antibodies were added to the wash buffer and incubated overnight at 4 °C on a rotator. The next day, samples were washed five times with wash buffer, then resuspended in pAG-MN buffer (wash buffer containing 120 ng/sample pAG-MN) and incubated for 30 minutes at 4 °C on a rotator. Following another five washes, digestion was initiated by resuspending the beads in wash buffer supplemented with 2 mM CaCl₂ and incubated at room temperature for 5 minutes, followed by an additional 25 minutes at 4 °C. The reaction was stopped by adding 250 mM EDTA/EGTA mix. Fragment release was initiated by the addition of 5 M NaCl and incubation at 37 °C for 30 minutes. The supernatant was collected, and beads were subsequently resuspended in 1× Urea buffer (100 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.5% IGEPAL, 8.5 M urea) for a second release step. After 30 minutes at room temperature, the urea release was combined with the initial supernatant. Samples were treated with SDS and proteinase K, followed by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. Library preparation was done using the KAPA Hyper Prep Kit for Illumina sequencing following the manufacturer's guidelines with modifications. 0.4× volume reactions were used for End repair and A-tailing steps, starting with 20 μl CUT&RUN DNA. The thermocycler conditions were 12°C for 15 min, 37°C for 15 min and 58°C for 25 min. Adapter ligation was also performed in 0.4× volume reactions. KAPA Dual Indexed adapters were used at 0.15 μM. A DNA cleanup was performed after ligation with Mag-Bind TotalPure NGS beads at 1.2× the sample volume, and DNA was resuspended in 10 mM Tris–HCl pH 7.5. Library amplification steps were performed in 0.5× volume reactions. The cycling conditions were: 45 sec initial denaturation at 98°C, 15 s denaturation at 98°C, 10 s annealing/elongation at 60°C, 1 min final extension at 72°C, hold at 4°C, for a total of 13 cycles. A post-amplification DNA purification was performed with beads at 1.2× sample volume. Libraries were then size-selected using the E-Gel EX 2% agarose gel and the E-Gel Power Snap Electrophoresis System. Run time was set to 10 min and DNA between 150 and 500 bp was excised from the gel and purified using the QIAquick Gel Extraction Kit according to the manufacturer's instructions. Libraries were quantified using Qubit  and the high sensitivity DNA kit. Libraries were pooled according to nM and sequenced with 36 bp pair-end reads on the NextSeq 550 using the Illumina NextSeq 500/550 High Output Kit v2.5 (75 cycles). Target depth was 5–10 million reads per sample.