title: M1 microglia disrupt the barrier function of retinal microvascular endothelial cells via ANGPT2/TIE2 axis
library_construction_protocol: 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.  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. 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.