GSM9741521: Day7 EC non-ischemic limb, WT, rep2; Mus musculus; RNA-Seq
Following skeletal muscle or lung tissue digestion, CD31+ ECs were isolated using CD31 MicroBeads (Miltenyi Biotec, 130-097-418) per the manufacturer's instructions. Briefly, cells were washed twice in cold, degassed buffer (PBS, 1% BSA, 2 mM EDTA) and centrifuged at 300g for 10 minutes to remove platelets. Up to 10⁷ cells were resuspended in 90 μL cold buffer, incubated with CD31 MicroBeads (10 μL per 10⁷ cells) for 15 minutes at 4°C, washed with 2 mL buffer, and resuspended in 500 μL buffer. Positive separation was performed on an autoMACS™ Pro Separator (Miltenyi Biotec); the magnetically retained fraction on MS Columns contained the CD31+ ECs. Total RNA quality was assessed on an Agilent TapeStation to determine RNA integrity numbers (RINs) before library preparation. mRNA was enriched with poly-T oligo-conjugated magnetic beads, fragmented, and reverse-transcribed to cDNA. During second-strand synthesis, dUTP was incorporated so that the second strand was not amplified, enabling strand-specific libraries. cDNA was end-repaired, ligated to indexed adapters, and PCR-amplified. After each step, nucleic acids were purified with AMPure XP beads (Beckman Coulter).