biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsLena EbertIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16734We generated full length V5-tagged WT JADE1, JADE2 and JADE3 constructs and the constructs lacking the PZP domains (JADE1_PZP, JADE2_PZP and JADE3_PZP) and expressed these constructs transiently in HEK293T cells. 24 hours after transfection the cells were harvest. Total RNA was extracted using the Direct-zol RNA Miniprep kit (Zymo Research) following the manufacturer’s instructions, including a DNase1 treatment. Afterwards we performed unbiased transcriptomic mRNA-seq analysis to analyze the importance of JADE1, JADE2 and JADE3 PZP mutants in transcriptional regulation.biostudies-arrayexpressLibrary Construction - 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.Sample Collection - 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).Nucleic Acid Extraction - 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.Sequencing - The pool was sequenced on an Illumina NovaSeq6000 with PE100 read length and a minimum of 35 million reads per sample.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesLena EbertfalseRNA-seq of JADE1/2/3 wildtype and PZP mutants in HEK293T cellWe generated full length V5-tagged WT JADE1, JADE2 and JADE3 constructs and the constructs lacking the PZP domains (JADE1_PZP, JADE2_PZP and JADE3_PZP) and expressed these constructs transiently in HEK293T cells. 24 hours after transfection the cells were harvest. Total RNA was extracted using the Direct-zol RNA Miniprep kit (Zymo Research) following the manufacturer’s instructions, including a DNase1 treatment. Afterwards we performed unbiased transcriptomic mRNA-seq analysis to analyze the importance of JADE1, JADE2 and JADE3 PZP mutants in transcriptional regulation.2026-05-15T00:00:00Z2026-05-15T07:52:11.52Z2026-03-11T22:44:35.428ZE-MTAB-16734ERP190691EFO_0002944EFO_0004170EFO_0005518EFO_0003738EFO_0004184