title: GSM9034677: Endothelial cells from mice cortex in ASD12h group, replicate 2 [ASD12h_2]; Mus musculus; RNA-Seq
library_construction_protocol: Upon completion of sleep deprivation, mouse cerebral cortex was rapidly dissected on ice. Tissue dissociation was performed using Type Ⅱ collagenase, followed by endothelial cell isolation via CD31+ magnetic bead sorting. mRNA was enriched using oligo(dT) magnetic beads for subsequent RNA sequencing. Total RNA serves as starting material. mRNA with polyA tails is enriched via Oligo(dT) magnetic beads. Enriched mRNA is fragmented in Fragmentation Buffer with divalent cations, then used as template for first-strand cDNA synthesis by M-MuLV reverse transcriptase with random primers. RNaseH degrades RNA strands, and DNA polymerase I synthesizes second-strand cDNA. Double-stranded cDNA is purified, end-repaired, A-tailed, and ligated with adapters. AMPure XP beads select 370–420bp fragments, which are PCR-amplified and purified to obtain high-quality libraries. For QC, Qubit 2.0 quantifies libraries (diluted to 1.5 ng/μl), Agilent 2100 checks insert sizes, and qRT-PCR ensures effective concentrations >2nM, meeting sequencing requirements.