GSM9110777: PC-12, control,rep2; Rattus norvegicus; RNA-Seq
Total RNA extraction using TRIZOL (15596-018, Ambion, USA). The RNA was treated with RQ1 DNase (Promega, USA) to remove DNA. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260nm/280nm (A260/A280) using Nanodrop One (Thermo, USA). RNA integrity was further verified by 1.5% agarose gel electrophoresis. For each sample, 1 μg of total RNA was used for RNA-seq library preparation by KAPA Stranded mRNA-Seq Kit for Illumina® Platforms (#KK8544, Roche, Switzerland). Polyadenylated mRNAs were purified by VAHTS mRNA capture Beads (N401-01, Vazyme, China) and Fragmented. Fragmented mRNAs were converted into double strand cDNA. Following end repair and A tailing, the DNAs were ligated to Diluted Roche Adaptor (KK8726). After purification of ligation product and size fractioning to 300-500bps, the ligated products were amplified, purified, quantified, and stored at -80℃ before sequencing. The strand marked with dUTP (the 2nd cDNA strand) was not amplified, allowing strand-specific sequencing. For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina NovaSeq 6000 system for 150 nt paired-end sequencing.