GSM9385001: D3 LPS 2; Mus musculus; RNA-Seq
The cells were lysed in 100 μL of TRIzol and total RNA was extracted with a miRNeasy Micro Kit (Qiagen). Bulk RNA sequencing was conducted on 4.5–7.5 × 10⁴ alveolar macrophages (AMs) that were pooled from 3–5 individual replicates for each condition. For samples yielding ≥ 100 ng of total RNA, libraries were conducted using the VAHTS Universal V8 RNA-seq Library Prep Kit (Illumina). Poly(A) mRNA was isolated using Oligo(dT) magnetic beads, fragmented via divalent cations and high temperature, and primed with random primers to synthesize first-strand and second-strand cDNA. The purified double-stranded cDNA underwent end repair and dA-tailing in a single reaction, followed by T-A ligation for adaptor attachment to both ends. Size selection of adaptor-ligated DNA was conducted using DNA purification beads, and each sample was PCR-amplified with P5 and P7 primers. Finally, the PCR products were subjected to quality validation.