biostudies-arrayexpressAlexis YatesHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15011This study was designed to investigate the consequences of substrate stiffness on primary brain microvascular endothelial cells (BMECs) in the presence of fluid shear stress (FSS) of 1.7 dyne/cm2. We utilized two gelatin (GEL) substrates of different w/v% (6.5% and 15%) that were crosslinked with microbial transglutaminase resulting in a Young's modulus of 6 kPa and 30 kPa (respectively).biostudies-arrayexpressGrowth Protocol - Primary human BMECs were purchased from Cell Systems (ACBRI 376) and maintained in Endothelial Cell Growth Media (C-22110, Sigma) up to passage 9. GEL substrates were coated with 50 ug/mL human collagen IV (C5533, Sigma) and 25 ug/mL bovine fibronectin (F1141, Sigma) for 24 hours before cell subculture. BMECs were subcultured on coated GELs either in glass plates (static condition) or in ibidi devices (u-Slide I Luer 3D, ibidi) for the FSS condition. All cells were subcultured at a density of 1e6 cells/mL and allowed to adhere for 24 hours before beginning FSS experiments. BMECs in ibidi devices were subjected to 1.7 dyne/cm2 FSS for 48 hours (following initial 24 hours of flow conditioning at 0.2 dyne) in endothelial cell media supplemented with 3% (w/v) 70kDa dextran and 1X penicillin-streptomycin. Static cultures were maintained in glass plates for 48 hours, with identical media supplements.Sample Collection - Following static or FSS culture, devices were removed from perfusion. Media was aspirated, cells were kept on GEL substrates and washed with PBS and immediately incubated with TRIzol for RNA extraction.Sequencing - NGS sequencing was performed via paired-end 150 bp on an Illumina NovaSeq X Plus with a targeted 50 million reads per sample.Library Construction - Purified RNA samples were submitted to the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core for bulk sequencing. cDNA libraries were prepared with a stranded mRNA (polyA-selected) library preparation kit.Nucleic Acid Extraction - At the end of each experiment, cells were lysed with TRIzol and RNA was extracted using a Direct-zol RNA miniprep kit (R2050, Zymo).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Demultiplexed FASTQ files were aligned using quasi-mapping transcriptome alignment with Salmon. A decoy-aware whole genome transcriptome index for alignment was created with the GENCODE GRCh28 primary assembly genome and v46 transcripts (GRCh38.primary_assembly.genome.fa, gencode.v46.transcripts.fa). Following quantification of paired-end reads with Salmon, count data was analyzed following the Bioconductor pipeline in R (version 4.4.2). Transcriptomic count data was imported and converted to gene-level quantification with tximeta28 and differential gene expression was quantified with the DESeq2 and results were compared for select conditions using a grouping variable (e.g. by FSS or hydrogel). Additional packages used: glmpca, paletteer, tidyr, apeglm, magrittr, dplyr, tidyverse.Data Transformation - The dataset was pre-filtered to remove genes with 0 or very low counts (< 10).UnknownTranscriptomicsGenomicsProteomicsN/AIllumina NovaSeq 6000Illumina Dragen RNAseq pipelineRNA-seq of coding RNAHomo sapiensEthan LippmannAlexis YatesfalseRNA-seq of primary human brain endothelial cells cultured on hydrogel substrates and exposed to static or fluid shear stress conditionsThis study was designed to investigate the consequences of substrate stiffness on primary brain microvascular endothelial cells (BMECs) in the presence of fluid shear stress (FSS) of 1.7 dyne/cm2. We utilized two gelatin (GEL) substrates of different w/v% (6.5% and 15%) that were crosslinked with microbial transglutaminase resulting in a Young's modulus of 6 kPa and 30 kPa (respectively).2025-12-01T00:00:00Z2025-12-01T02:01:45.91Z2025-04-04T11:39:06.84ZE-MTAB-15011ERP171272EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184