GSM9336892: brain,chronic stress,23 months of age,Model3; Mus musculus; RNA-Seq
After extracting total RNA from the target tissue or cells using the Trizol method, the integrity and purity of RNA are strictly detected (e.g., via agarose gel electrophoresis, Nanodrop, Qubit, and capillary electrophoresis) to ensure they meet the requirements for library construction. 1 μg total RNA was used for the following library preparation. For mRNA enrichment, poly(A) mRNA isolation was performed using Oligo(dT) beads. Alternatively, when the sample quality or experimental design necessitated, ribosomal RNA was depleted using rRNA removal methods to enrich for coding and non-polyadenylated transcripts. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA synthesis was carried out using reverse transcriptase, followed by second-strand cDNA synthesis. For strand-specific library construction, during the second-strand synthesis the standard dTTP was replaced with dUTP; subsequent treatment with USER enzyme selectively degrades the second strand prior to PCR amplification, thereby preserving strand information. The purified double-stranded cDNA was then treated to repair both ends and add a dA-tail in a single reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of adaptor-ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers, and the PCR products were validated. Finally, libraries with different indexes were multiplexed and loaded on an Illumina HiSeq/ Illumina Novaseq/ MGI2000 instrument for sequencing using a 2×150 paired-end (PE) configuration according to the manufacturer's instructions.