2025-02-02
ENA first public
ERC000011
ENA-CHECKLIST
Gehring Lab Institute for Genetics  University of Cologne
INSDC center name
public
INSDC status
ERS18330954
SRA accession
E-MTAB-13829_2:A006850321_209278
Submitter Id
ArrayExpress
broker name
Flp-In-T-REx-293
cell_line
epithelial cell
cell_type
not collected
collection date
human
common name
Protocols: Cells were harvested and lysed by adding 1 ml of in-house prepared TRI reagent to each well (prepared following DOI: 10.1371/journal.pbio.3000107). Both control and FKBP-UPF1-tagged HCT116 cell lines were maintained at 37°C and 5% CO2 in a humidified incubator in McCoy's 5A (Modified) Medium with GlutaMAX supplement (Gibco; Cat# 36600088), supplemented with 9% fetal bovine serum (Gibco; Cat# 10270106) and 1x Penicillin-Streptomycin (Gibco; Cat# 15140122). For control and FKBP-UPF1-tagged HEK293 cell lines, DMEM with high glucose and GlutaMAX supplement (Gibco; Cat# 61965059) was used and supplemented as described above. 5x10^5 cells were seeded in 6-well plates one day before starting the depletion experiment. UPF1 depletion was induced with 0.25 µM dTAGV-1 (Tocris Bioscience; Cat# 6914) 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. Libraries were prepared from 500ng total RNA with the Illumina® Stranded mRNA Preparation kit. ERCC RNA Spike-In Mix (Thermo Fischer) was added to the samples before library preparation. After poly-A selection (using Oligo(dT) magnetic beads), mRNA was purified, fragmented and reverse transcribed with random hexamer primers. Second strand synthesis with dUTPs was followed by A-tailing, adapter ligation and library amplification (12 cycles) to create the final cDNA libraries. After library validation and quantification (Agilent Tape Station), equimolar amounts of library were pooled.
description
late embryonic stage
developmental stage
normal
disease
CRISPR/Cas9
genetic modification
pCRIS-PITChv2-PurR-FKBP (UPF1 N)
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
kidney
organism part
1
replicate
Homo sapiens
scientific_name
female
sex
ERCC
spike in
A006850321_209278
title