Pcr primer mix

Trizol reagent (Invitrogen Life Technologies, Waltham, MA, USA) was used to isolate total RNA, and then NanoDrop (Thermo, Waltham, MA, USA) was used to determine RNA concentration, quality, and integrity. We used 3μgRNA to build a sequencing library according to the following steps. First, we used poly-T oligosaccharide-linked magnetic beads to purify mRNA from total RNA. In Illumina’s proprietary lysis buffer, divalent cations are used for lysis at high temperatures. We used random oligonucleotides and Super Script II to synthesize first-strand cDNA. Subsequently, DNA polymerase I and RNase H were used for second-strand cDNA synthesis. In order to select cDNA fragments of 400–500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter). The DNA fragments with linker molecules at both ends were subjected to a 15-cycle PCR reaction using the Illumina PCR primer mix. The product was purified (AMPure XP system) and quantified using Agilent’s high-sensitivity DNA analysis on the Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on the NovaSeq 6000 platform (Illumina) by Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).

C2C12 cells cultured in the growth medium were treated without (control) or with 50 µM LA and AA. Then, total RNA was isolated using the Trizol Reagent (Biosharp, Beijing, China), after which the concentration, quality, and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). Then, sequencing libraries were generated using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). The library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA) to select cDNA fragments of the preferred 200 bp length, and DNA fragments with ligated adaptor molecules on both ends were selectively enriched using an Illumina PCR Primer mix in a 15-cycle PCR reaction. Then, products were purified (AMPure XP system) and quantified using the Agilent high-sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA). The sequencing library was then sequenced on a Hiseq platform (Illumina) by Shanghai Personal Biotechnology Co. Ltd. (Shanghai, China).