Vahtstm dna clean beads

Total RNAs was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After extracted, total RNAs were treated with RNase R to degrade the linear RNAs, and purified using RNeasy MinElute Cleanup Kit (Qiagen,Venlo, The Netherlands). Next, strand-specific library was constructed using VAHTS Total RNAseq (H/M/R) Library Prep Kit (Vazyme, Nanjing, China) for Illumina following the manufacture’s instructions. Briefly, ribosome RNAs were removed to retain circRNAs. The enriched circRNAs were fragmented into short fragments by using fragmentation buffer and reverse transcribed into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP (dUTP instead of dTTP) and buffer. Next, the cDNA fragments were purified with VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China), end repaired, A base added, and ligated to Illumina sequencing adapters. Then UNG (Uracil-N-Glycosylase) was used to digest the second-strand cDNA. The digested products were purified with VAHTSTM DNA Clean Beads, PCR amplified, and sequenced using Illumina Novaseq6,000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

Total RNA was extracted from LDM tissues with Trizol reagent kit (Invitrogen, Carlsbad, CA, United States) according to the instructions. RNA quality was detected on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, United States), and checked with RNase free agarose gel electrophoresis. Then total RNA was treated with RNase R to degrade the linear RNAs, and purified with the RNeasy MinElute Cleanup kit (Qiagen, Venlo, Netherlands). Strand-specific library was constructed with VAHTS Total RNAseq (H/M/R) Library Prep Kit (Vazyme, Nanjing, China) for Illumina. In a word, ribosome RNAs were removed to retain circRNAs.
The enriched circRNAs were fragmented into short fragments with fragmentation buffer and transcribed into cDNA with random primers. Second-strand were synthesized with DNA polymerase I, RNase H, dUTP and buffer. And then, the cDNA fragments were purified with VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. Then Uracil-N-Glycosylase (UNG) was used to digest the second-strand cDNA. The digested products were purified with VAHTSTM DNA Clean Beads, PCR amplified, and sequenced by Illumina Novaseq60000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

Eight total RNA samples were obtained to generate RNA libraries for each sample. After the samples were qualified, using mRNA Capture Beads Enriched eukaryote mRNA, mRNA was fragmented by heating. These short mRNA and random hexamers were used to generate the first cDNA and then the second cDNA was synthesized. The second cDNA was purified using VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and then ligated to sequencing adapters. The fragments were amplified by using polymerase chain reaction (PCR) and purified using VAHTSTM DNA Clean Beads and then sequenced using an Illumina HiSeq (Vazyme, Nanjing, China). Raw sequence data were assessed, and sequences containing adaptor tags and those of low quality were excluded. Filtered reads were used for subsequent analysis, and the unique reads were used to identify differentially expressed genes (DEGs) with |log2Ratio|≥ 1 and q-value |FDR|≤ 0.05.