biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsXiao FengIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15209Human microglial clone 3 cells (HMC3) was induced to M1 or M2 status, then co-cultured with retinal microvascular endothelial cells (hRMECs) in vitro.biostudies-arrayexpressNucleic Acid Extraction - Isolated RNA was confirmed RNA purity (A260/A280 ratio should be ~2.0) and integrity (RIN > 7) to ensure high-quality starting material. Use VAHTS mRNA Capture Beads (Vazyme) to capture polyadenylated mRNA, leaving out other RNA types. Convert the enriched or depleted RNA to complementary DNA (cDNA) through reverse transcription. Perform first-strand synthesis using reverse transcriptase and random primers. Follow with second-strand synthesis to create double-stranded cDNA.Library Construction - Perform first-strand synthesis using reverse transcriptase and random primers. Follow with second-strand synthesis to create double-stranded cDNA. Repair the ends of cDNA fragments and add a single \"A\" nucleotide at the 3' end to prevent self-ligation.Sample Collection - Human microglial clone 3 (HMC3) cells (Cat. No. CRL-3304) and primary renal mixed epithelial cells (hRMECs) (Cat. No. PCS-400-012) were purchased from ATCC. HMC3 cells were treated with 1 μg/mL LPS (Cat. No. HY-D1056, MCE, China) or 20 ng/mL IL-4 (Cat. No. HY-P70445, MCE) for 24 hs. HMC3 and hRMECs were co-cultured using Corning 12 mm Transwell with 0.4 μm pore polycarbonate membrane insert (Cat. No. 3401, Corning, USA) for 48 hs after induction.Sequencing - Ligate sequencing adapters to the cDNA fragments for compatibility with the Illumina sequencing platform NovaSeq 6000. Purify and size-select the 300 bp cDNA fragments to ensure uniform fragment sizes. Amplify the cDNA library using PCR to increase the number of fragments for sequencing, ensuring the library concentration is sufficient. Verify the size distribution and concentration of the library using a bioanalyzer or similar device. Ensure that the library is free of adapter dimers or contaminants and falls within the desired size range. Load the library onto the sequencing platform according to the manufacturer's instructions. Set the 150 bp paired-end reads and 10 M sequencing depth.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesXiao FengfalseM1 microglia disrupt the barrier function of retinal microvascular endothelial cells via ANGPT2/TIE2 axisHuman microglial clone 3 cells (HMC3) was induced to M1 or M2 status, then co-cultured with retinal microvascular endothelial cells (hRMECs) in vitro.2025-06-20T00:00:00Z2025-06-05T12:19:03.159Z2025-06-05T12:19:03.159ZE-MTAB-15209ERP173192EFO_0002944EFO_0004170EFO_0005518EFO_0003738EFO_0004184