HCT_N_AID_UPF1_0h_IAA_ONT_dRNA_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. Poly(A) mRNA was purified from total RNA using the Dynabeads™ mRNA Purification Kit (Invitrogen; Cat#61006). After one round of purification according to the manufacturer's instruction, the samples were incubated for a second round of binding to the same beads, repeating the protocol for a second time after the rebinding of the RNA to the beads. Successful enrichment for poly(A) RNA was confirmed by High Sensitivity RNA ScreenTape Analysis. ONT direct RNA sequencing libraries were prepared from 300 ng of poly(A) RNA using the Direct RNA Sequencing Kit (Oxford Nanopore Technologies, #SQK-RNA004) following the manufacturer's instructions.
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_ONT_dRNA_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_ONT_dRNA_2
scientific_name
Homo sapiens
sex
male