2025-02-02
ENA first public
ERC000011
ENA-CHECKLIST
Gehring Lab Institute for Genetics  University of Cologne
INSDC center name
public
INSDC status
ERS18230349
SRA accession
E-MTAB-13794:K002000214_84657
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 with 350 µl lysis buffer from the NucleoSpin RNA Plus kit (Macherey&Nagel; Cat# 740984.50). The HEK293 cell line was maintained at 37°C and 5% CO2 in a humidified incubator DMEM with high glucose and GlutaMAX supplement (Gibco; Cat# 61965059), supplemented with 9% fetal bovine serum (Gibco; Cat# 10270106) and 1x Penicillin-Streptomycin (Gibco; Cat# 15140122). 2x10^5 cells were seeded in 6-well plates and reverse transfected with 2.5 µl Lipofectamine RNAiMAX (Invitrogen; Cat# 13778150) and 60 pmol of the respective siRNAs (Luciferase- or EIF4A3-targeting) according to the manufacturer’s protocol. Medium was changed the next day and cells were harvested 3 days after the initial knockdown. Total RNA was extracted with the NucleoSpin RNA Plus kit (Macherey-Nagel; Cat# 740984.50) according to the manufacturer's instructions. The Lexogen SIRV Set1 Spike-In Control Mix that provides a set of external RNA controls was added to the total RNA to enable performance assessment. Using the Illumina TrueSeq Stranded Total RNA kit library preparation was accomplished. This included removal of ribosomal RNA via biotinylated target-specific oligos combined with Ribo-Zero gold rRNA removal beads from 1 μg total RNA input. After depletion of cytoplasmic and mitochondrial rRNA, the remaining RNA was purified and fragmented using divalent cations under elevated temperature. The RNA fragments underwent reverse transcription using random primers. This was followed by second strand cDNA synthesis with DNA Polymerase I and RNase H. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified to create the final cDNA libraries. Next library validation and quantification (Agilent tape station) were performed, followed by pooling of equimolar amounts of library.
description
late embryonic stage
developmental stage
normal
disease
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
kidney
organism part
2
replicate
Homo sapiens
scientific_name
female
sex
SIRV-Set 1: E0
spike in
K002000214_84657
title