HCT_N_AID_UPF1_0h_IAA_PacBio_2
Homo sapiens
Protocols: Cells were harvested and lysed by adding 2 ml Invitrogen™ TRIzol™ Reagent (Invitrogen; Cat# 15596018) to each plate. The N-AID-UPF1-tagged HCT116 cell line was maintained at 37°C and 5% CO2 in a humidified incubator in McCoy's 5A (Modified) Medium (Gibco), supplemented with 10% fetal bovine serum (Gibco) and 2 mM L-glutamine (Gibco). 5x10^6 cells were seeded per 15 cm dish one day before starting the depletion experiment. UPF1 depletion was induced with 500 µM indole-3-acetic acid (IAA; Sigma-Aldrich; Cat# I5148) for 12 hours. All cells were harvested at the same time to minimize differences in cell numbers. Total RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research; Cat# R2052) including the recommended DNase I treatment according to the manufacturer's instructions.  The PacBio Kinnex full-length RNA sequencing library was constructed from four total RNA samples. First, each sample was processed into a PacBio barcoded Iso-Seq library, starting with 420 ng of total RNA and using the Iso-Seq Express 2.0 Kit (PacBio, #103-071-500). Then, 14 ng of each Iso-Seq library was additionally amplified to introduce Kinnex overhangs at the ends of the library molecules and pooled together to prepare one Kinnex library using the Kinnex Concatenation Kit (PacBio, #103-072-000).
ENA first public
2025-09-25
INSDC center name
Gehring Lab Institute for Genetics University of Cologne
INSDC status
public
Submitter Id
E-MTAB-14725_2:HCT_N_AID_UPF1_0h_IAA_PacBio_2
broker name
ArrayExpress
cell line
HCT116
cell type
colorectal cancer cell
collection date
not collected
common name
human
developmental stage
adult
disease
colorectal adenocarcinoma
genetic modification
CRISPR/Cas9
genotype
pSH-EFIRES-P-AtAFB2-mCherry UPF1-nterm BSD/Hygro P2A V5 AID7 (U1A3n2)
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
colon
replicate
2
sample name
E-MTAB-14725_2:HCT_N_AID_UPF1_0h_IAA_PacBio_2
scientific_name
Homo sapiens
sex
male