Sample 19
Homo sapiens
Protocols: Full length JADE1, JADE2 and JADE3 cDNAs were amplified by PCR from human embryonic kidney (HEK) 293T cell cDNAand were cloned into a modified pcDNA6 vector (Invitrogen) containing an N-terminal V5-tag using standard molecular cloning techniques. The JADE1_PZP mutant lacks amino acids (aa) 199-373, the JADE2_PZP mutant lacks aa 195-369, and the JADE3_PZP mutant lacks aa 196-370 were generated via Gisbon assembly. The plasmid containing EPS1-225 cDNA has been previously described. HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS. For transfection HEK293T cells were seeded in 10 cm dishes and grown to 50-60% confluency. Plasmid DNA was transiently transfected using the calcium phosphate method. 48 h after transfection cells were harvested in TRI Reagent® (Zymo Research). Total RNA was extracted using the Direct-zol RNA Miniprep kit (Zymo Research) following the manufacturer's instructions, including a DNase1 treatment. The pellet was resuspended in 30 μl of RNase-free water and RNA yield and quality were assessed using a Nanodrop 1000 spectrophotometer.  Library preparation and sequencing was performed by the Cologne Center for Genomics (CCG). In brief, libraries were prepared using the Illumina® TruSeq® mRNA stranded sample preparation Kit. Following poly-A selection, mRNA was purified and fragmented using divalent cations at elevated temperature. Fragmented RNA was reverse-transcribed using random primers, 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. Library quality and concentration were assesed using a TapStation 4200 (Agilent). Equimolar amounts of libraries were pooled and quantified by using the KAPA Library Quantification Kit (Peqlab) on an Applied Biosystems 7900HT Sequence Detection System.
ENA first public
2026-05-16
INSDC center name
Department II of Internal Medicine and Center for Molecular Medicine Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
INSDC status
public
Submitter Id
E-MTAB-16734:Sample 19
broker name
ArrayExpress
cell line
HEK293T
collection date
not collected
common name
human
developmental stage
embryonic stage
genotype
CON
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
kidney
replicate
3
sample name
E-MTAB-16734:Sample 19
scientific_name
Homo sapiens