GSM5242518: CDK8_pt_NT_0H-2; Homo sapiens; OTHER
Cells from one 10-cm dish containing ~8 × 106 HCT116 cells were transferred to a 1.5ml tube, and cell numbers were measured. Cells were cross-linked with 0.1% formaldehyde in phosphate-buffered saline for 2 min at room temperature before quenching with glycine to a final concentration of 0.125 M. Cells were resuspended in NE buffer, incubated on ice for 10 min, and nuclei were collected centrifugation at 600xg for 5 min. Cut&Tag assays were performed with up to 100,000 nuclei according to the method of Kaya-Okur et al. (2020). All libraries were sequenced on an Illumina NextSeq 500 according to the manufacturer's instructions. To amplify libraries as previously described in  Kaya-Okur et al. (2020), sample was mixed with 2 μL of a universal i5(10 μM) and a uniquely barcoded i7 primer((10 μM), using a different barcode for each sample. A volume of 25 μL NEBNext HiFi 2× PCR Master mix was added and mixed. The sample was placed in a Thermocycler with a heated lid using the following cyclingconditions: 72 °C for 5 min (gapfilling); 98 °C for 30 s; 14 cycles of 98 °C for 10 sand 63 °C for 30 s;final extension at 72 °C for 1 min and hold at 8 °C. Post-PCR clean-up was performed by adding 1.3× volume of Ampure XP beads (BeckmanCounter), and libraries were incubated with beads for 15 min at RT, washed twicegently in 80% ethanol, and eluted in 30 μL 10 mM Tris pH 8.0.
GEO Accession
GSM5242518