GSM9757442: Lenti-shEHMT1-1; Homo sapiens; RNA-Seq
Human aortic smooth muscle cells (HASMCs) cultured in 10 cm dishes were infected with Lenti-shRNA (control) or Lenti-shEHMT1, with three replicates per group. After culture, cells were harvested using Trizol, and total RNA was extracted by phenol-chloroform extraction. RNA concentration and integrity were precisely analyzed using an Agilent 2100 Bioanalyzer. Total RNA (≥1 μg) was used for library construction. mRNA with polyA tails was enriched using Oligo(dT) magnetic beads and then randomly fragmented with divalent cations in Fragmentation Buffer.  2) The fragmented mRNA was used as template, and random oligonucleotides as primers, to synthesize first‑strand cDNA in a reverse transcriptase (M‑MuLV) system, followed by mRNA degradation with RNase H.  3) Second‑strand cDNA was synthesized in a DNA polymerase I system using the first‑strand cDNA as template and dNTPs as substrates.  4) The purified double‑stranded cDNA was subjected to end repair, polyA tailing, and adapter ligation. DNA fragments of 370–420 bp were selected using magnetic beads, and the library was amplified by PCR.  5) After library preparation, preliminary quantification was performed using a Qubit 2.0 Fluorometer, followed by quality assessment on an Agilent 2100 Bioanalyzer. Finally, the library was accurately quantified by real‑time PCR to ensure it met the quality requirements for sequencing.  c) Sequencing on the instrument: After passing quality control, the libraries were sequenced according to the required concentration and data output. Sequencing was performed using the principle of sequencing‑by‑synthesis. DNA polymerase, four fluorescently labeled dNTPs, and adapter primers were added to the reaction system for amplification. Because different dNTPs carry distinct fluorescent labels, the fluorescence signals were captured by the sequencer and converted into corresponding base calls by a computer. After sequencing was completed, the reads were assembled to generate fragment information