GSM9151961: AKTP MTO93 mouse tumor organoids, RM-044, JunB, replicate 2; Mus musculus; OTHER
Organoids were dissociated for 30 min with TryplE at 37ºC. Cells were washed twice in wash buffer for 3 min at 600 g, RT. Aliquots of 100,000 cells were prepared. Cells were then bound to Concavalin A-coated magnetic beads (BioMag®Plus, 86057-3) and permeabilised by resuspension in antibody buffer (wash buffer with 0.05% digitonin) and the appropriate dilution of antibody (H3K27ac, JunB, IgG). Samples were incubated overnight at 4ºC o/n a rocking platform. Next day, beads were resuspended in the digitonin-wash buffer containing pAG-MNase (Cell Signaling, 0366S)­. Samples were then incubated on a rocking platform for 1h at 4ºC. Beads were resuspended in digitonin-wash buffer and placed in an ice water bath (0 ºC) for 5 min. Chromatin digestion was performed by adding CaCl2 and incubating for 30 min at 4 ºC. After digestion, 2x STOP buffer was added and samples were incubated for 10 min at 37ºC. Tubes were centrifuged for 5 min at 16,000 g, 4 ºC, and placed on a magnetic rack. The supernatant containing the digested chromatin was purified using the Qiagen MinElute PCR Purification Kit (QIAGEN, 28006). DNA was eluted in 25 μl of Nuclease-Free Water, quantified with Qubit HS dsDNA (ThermoFisher, Q33231), and fragment size distribution was assessed using a D5000 ScreenTape (Agilent, 5067-5588) for the 4150 TapeStation system. Libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645L) without size selection before PCR amplification. Amplification was performed with Q5 Master Mix, Unique Dual Index Primer Pairs (NEB, E6440S), and 20 μl of library. The PCR program was the following: 45 sec at 98 ºC, 14 cycles consisting of 15 sec at 98 ºC and 10 sec at 60 ºC and a final extension of 1 min at 72 ºC. DNA purification was done in 1.1x volume AMPure XP beads (BECKMAN COULTER, 082A63881). Library concentration and size were assessed with Qubit HS dsDNA (ThermoFisher, Q33231) and D5000 ScreenTape (Agilent, 5067-5588) for the 4150 TapeStation system.