GSM8679824: 2-3 (LPS group); Homo sapiens; RNA-Seq
Total RNA was isolated using TRIZOL reagent (Ambion, Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Each group was prepared for triplicate. These samples were shipped on dry ice and a secure foam box, within 48 hours to the sequence company (GENESKY, Shanghai, China). Total RNA extraction from the sample was performed using the TruSeq RNA sample Preparation kit V2(Illumina, USA). Total RNA concentration and quality were determined using an Invitrogen Qubit 3.0 Spectrophotometer (Thermo Fisher Scientifific, USA). Before library construction, RNA integrity was confirmed by agarose gel electrophoresis (Agilent Bioanalyzer 2100; Agilent Technologies), to ensure that the RNA Integrity Number (RIN) values were ≥6.5. Purify mRNA from total RNA using oligo magnetic poly-α beads and separate into fragments of 100–300 bp. Using RNA as a template, use 6-base random primers and reverse transcriptase to synthesize the first-strand DNA, and use the first-strand cDNA as a template to synthesize the second-strand cDNA. During second-strand cDNA synthesis, the base T is replaced by U, resulting in a strand-specific library. RNA-Seq is carried out using the TruSeq RNA sample preparation kit V2 (Illumina, USA), and strand-specific libraries were used to determine the transcriptional orientation of both sense and antisense strands to improve the accuracy of subsequent annotation of gene function and analysis of gene expression. After building the library, enriched library fragments were PCR amplified and screened based on fragment size to yield a 300 to 400 bp library. The total and effective concentrations of the libraries were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA).