title: Sample 2
organism: Homo sapiens
description: Protocols: 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.
attributes.tag: ENA first public
attributes.value: 2024-06-15
attributes.tag: External Id
attributes.value: SAMEA114785906
attributes.tag: INSDC center name
attributes.value: Center for Omics Sciences IRCCS San Raffaele Scientific Institute
attributes.tag: INSDC last update
attributes.value: 2023-12-03T10:34:20Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-13597:Sample 2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: HT-29
attributes.tag: cell_type
attributes.value: epithelial cell
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: colorectal cancer
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: colon
attributes.tag: sample name
attributes.value: E-MTAB-13597:Sample 2
attributes.tag: scientific_name
attributes.value: Homo sapiens