title: GSM9308551: Cardiac tissue from AB with LL-VNS 20min starting 4w biological replicate 1; Mus musculus; RNA-Seq
library_construction_protocol: At 8 weeks post-AB surgery, mice were euthanized by CO2 asphyxiation followed by cervical dislocation. Hearts were rapidly excised, and left ventricular tissue was dissected, snap-frozen in liquid nitrogen, and stored at -80°C until RNA extraction. Total RNA was extracted from left ventricular tissue using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA quality was assessed using Agilent 2100 Bioanalyzer with RNA integrity numbers (RIN) greater than 8.0 required for library construction. RNA concentration was measured using NanoDrop spectrophotometry. RNA library construction was performed using the VAHTS mRNA-seq V3 Library Prep Kit for Illumina (Vazyme Biotech) starting from 1 μg total RNA per sample. The protocol included: (1) PolyA RNA isolation using oligo-dT magnetic beads, (2) RNA fragmentation to approximately 200-300 bp, (3) First-strand cDNA synthesis using random hexamer primers, (4) Second-strand cDNA synthesis, (5) End repair and A-tailing, (6) Adapter ligation, (7) Size selection using AMPure XP beads, (8) PCR amplification (12-15 cycles), (9) Final library quality control using Agilent 2100 Bioanalyzer. Libraries were quantified using Qubit fluorometry and qPCR, pooled at equimolar ratios, and validated for appropriate insert size distribution.Paired-end sequencing (2×150 bp) was performed on the Illumina NovaSeq 6000 platform. Libraries were loaded onto S4 flow cells with target sequencing depth of >40 million read pairs per sample to ensure adequate coverage for differential expression analysis. Sequencing was performed by Wuhan IGENEBOOK Biotechnology Co., Ltd.