CUT&Tag profiling of HNF4A in HT29 cell lines after the depletion of PINK1
CUT&Tag profiling of HNF4A in HT29 cell lines after the depletion of PINK1
Cells were trypsinized, harvested and washed with PBS 1%. Then, the pellet was immediately used for RNA extraction or frozen and stocked at -80°C until use. Cells were grown in DMEM supplemented with 10% Fetal Bovine Serum. About 1.2e6 HT29 cells were plated on 6 cm plates. The day after cells were overnight infected with SCR or shPINK1 constructs. After 24h, Puromycin was added at 2ug/ml concentration for each plate. After three days of selection, cells were harvested for CUT&Tag experiment and sequencing. Tagmentation was performed according to the manufacturer guidelines. The experiment was performed mainly following the protocol: CUT&Tag-direct with CUTAC V.3 available at https://www.protocols.io (dx.doi.org/10.17504/protocols.io.bqwvmxe6). In particular, we started from SCR and shPINK1 HT29 samples and we extracted and slightly fixed nuclei according to the protocol-appendix. A total amount of 100.000 nuclei for each reaction were prepared as starting material. The primary antibody (HNF4α, C11F12, Rabbit mAb #3113, Cell Signaling technology®️, 1µg/mL) incubation was performed over-night and a one-hour incubation with the secondary antibody (Guinea Pig anti-Rabbit IgG, ABIN101961, antibodies-online.com) followed. Both immunoreactions were performed in a 100µL volume. To ensure Tn5 binding, samples were incubated with the CUTANA™ pAG-Tn5 (15-1017, Epicypher®️, 20X dilution) for 2 hours at room temperature in a 50µL volume.