title: GSM9002239: WT Control organoids CUT&RUN with IgG; Mus musculus; OTHER
library_construction_protocol: Mouse squamous organoids were mechanically dissociated from Matrigel, dispersed using 0.05% Trypsin-EDTA (Thermo Fisher) and manual pipetting. 400,000 cells were used per condition according to the CUTANA CUT&RUN Kit (EpiCypher). Briefly, ConA beads were activated and dissociated cells were bound to beads and incubated with antibodies (0.1 µg H3K27me3, MA5-11198, Invitrogen; 0.5 µg SOX2, ab97959, Abcam; IgG 0.5 µg, 13-0042, EpiCypher) on nutator overnight at 4°C. The slurry was washed with cold digitonin (0.01%) buffer prior to binding of pAG-MNase (EpiCypher) and incubation for 10 minutes at room temperature. 100 mM of CaCl2 was added to activate pAG-MNase for 2 hours at 4°C prior to stop buffer chelating of calcium and DNA purification with the CUTANA DNA Purification Kit. Illumina NGS libraries were prepared using 5 ng of DNA from each condition and the CUTANA CUT&RUN Library Prep Kit. Agilent TapeStation was used to calculate molarity of DNA fragments. DNA libraries were pooled for a final concentration of 10 nM.