Dna clean beads

Total RNA was extracted from AAA tissues (N = 3) and normal abdominal aortic tissues (N = 3) with a Trizol RNA extraction reagent (Sigma–Aldrich, Shanghai, China). The quantity and purity of RNA were detected by the NanoDrop spectrophotometer (Thermo Fisher Scientific Inc., Shanghai, China). The RNA was electrophoresed on 1.2% agarose gel to analyze the integrity. For RNA-seq library construction, 1 μg of total RNA was used. The RNA libraries were constructed using the Total RNA-seq (HMR) Library Prep Kit (Azyme Biotech Co., Ltd., Nanjing, China). In brief, ribosomal RNA was removed with rRNA probes and RNase H, RNA was fragmented to ∼300 bp by metal ion, reverse transcribed into cDNA, and connected with adapters. The fragments were sorted by DNA Clean Beads (Beckman, USA), and subjected to PCR amplification and purification and libraries were validated using Agilent 2200 (Agilent, Santa Clara, CA, USA). Illumina platform (Illumina, Inc., CA, USA) was used for RNA sequencing.

To assess the RNA degradation and contamination, 1% agarose gel electrophoresis was conducted, and the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) was used to test the RNA purity. RNA integrity was assessed by using the Bioanalyzer 2100 System (Agilent Technologies, CA, USA).
For RNA-sequencing of 13 paired samples, mRNA Capture Beads with Oligo(dT) (VAHTS, Nanjing, China) were first used to capture mRNA. mRNA was then purified with binding and washing buffer. mRNA was then randomly fragmented and reverse transcribed into cDNA in the corresponding buffer. Random primers were used to synthesize the first cDNA strand, and dNTPs/DNA polymerase I (ABclonal, MA, USA) was used to synthesize the second cDNA strand. After dA—tailing of 3' DNA fragments, UMI (Novogene, Beijing, China) and sequence adapter were used to ligate DNA. After PCR, the product was purified and collected using DNA Clean Beads (Beckman Coulter, CA, USA) and nuclease-free H₂O. Each analysis was carried out in triplicate. After quality assessment, the prepared library was sequenced on the Illumina platform (Illumina Novaseq, Inc., USA).