title: GSM6865780: KPC1 PDAC tumor cells treated with T/P in vitro [KPC1 TP1]; Mus musculus; OTHER
library_construction_protocol: CUT&Tag was performed largely as described in Kaya-Okur et.al. (Nature Protocols, 2020). FACS sorted GFP+ tumor cells from transplanted PDAC tumors or trypsinized cells in culture were counted and 100,000 cells were washed and resuspended in wash buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail) and used for CUT&Tag. 10 µL of activated Concanavalin A coated magnetic beads (Polysciences) were added per sample and incubated at room temperature for 15 min. Bead-bound cells were resuspended in 100 µL Dig-wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 0.05% Digitonin; 1× Protease inhibitor cocktail) containing 2 mM EDTA and 1ul of H3K27me3 antibody (ThermoFisher, MA5-11198). The mixture was incubated overnight at 4 °C for antibodies to bind. After pulling beads to the side of the tube using a magnetic rack and removal of unbound primary antibody, beads were resuspended in 100ul Dig-wash Buffer containing 1ul of Guinea Pig anti-Rabbit antibody (Antibodies-Online ABIN101961) and incubated for 30 mins at RT. Cells were washed 3 times with Dig-wash and then incubated with a 1:50 dilution of pA-Tn5 adapter complex in Dig-med (0.05% Digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1X Protease inhibitor cocktail) at RT for 1 hr. Cells were washed thrice in Dig-med Buffer and then resuspended in 300 µL Dig-med Buffer containing 10 mM MgCl2 and incubated at 37 °C for 1 h to activate tagmentation. To stop tagmentation, 10 µL of 0.5 M EDTA, 3 µL of 10% SDS and 1 µL of 20 mg/mL Proteinase K was added to each tube, which were incubated at 55 °C for 1 hour. DNA was extracted by performing one phenol:chloroform extraction followed by ethanol precipitation. The DNA pellet was resuspended in 22 µL of 10 mM Tris pH 8. CUT&Tag libraries were amplified by mixing 21 µL of tagmented DNA with 2µL each of (10 µM) barcoded i5 and i7 primers, using a different combination for each sample. 25 µL NEBNext HiFi 2× PCR Master mix (NEB) was added, and PCR was performed using the following cycling conditions: 72 °C for 5 min (gap filling); 98 °C for 30 s; 17 cycles of 98 °C for 10 s and 63 °C for 30 s; final extension at 72 °C for 1 min and holding at 4 °C. 1.1× volumes of Ampure XP beads (Beckman Coulter) were incubated with libraries for 10 min room temperature to clean up the PCR reaction. Bead bound DNA was purified by washing twice with 80% ethanol and eluting in 20 µL 10 mM Tris pH 8.0.