2025-06-01
ENA first public
ERC000011
ENA-CHECKLIST
Gehring Lab Institute for Genetics  University of Cologne
INSDC center name
public
INSDC status
ERS22675790
SRA accession
E-MTAB-14725:HCT_N_AID_UPF1_12h_IAA_ONT_dRNA_1
Submitter Id
ArrayExpress
broker name
HCT116
cell_line
colorectal cancer cell
cell_type
not collected
collection date
human
common name
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.
description
adult
developmental stage
colorectal adenocarcinoma
disease
CRISPR/Cas9
genetic modification
pSH-EFIRES-P-AtAFB2-mCherry UPF1-nterm BSD/Hygro P2A V5 AID7 (U1A3n2)
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
colon
organism part
1
replicate
Homo sapiens
scientific_name
male
sex
HCT_N_AID_UPF1_12h_IAA_ONT_dRNA_1
title