GSM9615856: HIF-1α knockdown; Mus musculus; RNA-Seq
RNA from the total sample was isolated and purified using TRIzol (thermofisher) according to the manufacturer's operating protocol. The quantity and purity of the total RNA were then quality controlled using a NanoDrop ND-1000, and RNA integrity was detected using a Bioanalyzer 2100; concentration >50 ng/μL, RIN value >7.0, and total RNA >1 μg met the requirements for downstream experiments. Poly(A)+ mRNA was enriched from total RNA using oligo(dT) magnetic beads and fragmented at high temperature in the presence of divalent cations. The fragmented RNA was reverse transcribed into first-strand cDNA, followed by second-strand synthesis using DNA polymerase I and RNase H. During second-strand synthesis, dUTP was incorporated to generate strand-specific libraries. The double-stranded cDNA was end-repaired, A-tailed, and ligated to sequencing adapters. After size selection and purification, the second strand was digested using UDG, and the library was amplified by PCR. The final libraries (~300 bp insert size) were sequenced on the Illumina NovaSeq 6000 platform with paired-end 150 bp reads.