biostudies-arrayexpressClaudio RaimondiHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15027Investigate the effect of silencing ABCB8 on the transcriptional profile of HUVECsbiostudies-arrayexpressNucleic Acid Extraction - mRNA was collected using the Monarch® Total RNA Miniprep Kit (New England BioLabs, UK; cat#: T2010S) and cDNA was prepared using LunaScript® RT SuperMix Kit (New England BioLabs, UK; cat#: E3010L).Sample Collection - HUVECs transfected with siRNA targeting ABCB8 or with a non-targeting siRNA were cultured for 72 hours. RNAs from 4 independent experiments were isolated using the protocol described below and transcriptomic analysis performed by Novogene, UK.Library Construction - A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra TM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X) or by using sonication with Diagenode bioruptor Pico for breaking RNA strands. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.Sequencing - The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Gene read counts were calculated using FeatureCounts, and reads per kilobase of exon model per million mapped reads (RPKM) for each gene were calculated based on gene length and read counts. Differential expression analysis was conducted using the DESeq2 R package. P values were adjusted for false discovery rate (FDR) using the Benjamini and Hochberg method. Genes with an adjusted P value of <0.05 were considered differentially expressed. GO enrichment analysis of differentially expressed genes was performed using the clusterProfiler R package, with GO terms having a corrected P value of <0.05 deemed significantly enriched.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensClaudio RaimondifalseRNAseq of HUVECs transfected with siRNA targeting ABCB8 or a non-targeting control siRNAInvestigate the effect of silencing ABCB8 on the transcriptional profile of HUVECs2025-11-15T00:00:00Z2025-11-15T02:02:23.684Z2025-04-10T14:49:13.526ZE-MTAB-15027ERP171486EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184