Dna clean beads

Following extraction, total RNA was treated with RNase R to degrade the linear RNA and purified with RNeasy MinElute Cleanup Kit (Qiagen, Inc., Valencia, CA, USA). Next, a strand-specific library was constructed with VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina according to the manufacturer's protocol. In brief, ribosomal RNA was removed to retain the circRNAs. The enriched circRNAs were broken into short fragments using a fragmentation buffer and reverse transcribed into cDNA with random primers. Secondly, strand cDNA fragments synthesized by DNA polymerase I were purified with VAHTSTM DNA Clean Beads and liquated to Illumina sequencing adapters. Uracil-N-glycosylase was used to digest the second-strand cDNA. The digested products were purified with VAHTSTM DNA Clean Beads, amplified, and sequenced with Illumina HiSeq™ 2500 by Gene Denovo Biotechnology Co. (Guangzhou, China). The edgeR package (http://www.rproject.org/) was used to identify differentially expressed circRNAs. Some significant circRNAs were blasted against the circBase for annotation [13 (link)]. The circRNAs that could not be annotated were defined as novel circRNAs.

RNA‐seq libraries were prepared using 1 µg of total RNA. Poly‐adenylated RNA isolation, cDNA synthesis, end‐repair, ligation of the Illumina indexed adapters, and libraries amplification were finished by Fast RNA‐seq Lib Prep Kit V2. Libraries were selected for 200 – 300 bp cDNA fragments using VAHTS DNA Clean Beads and were sequenced on the Illumina NovaSeq 6000 v1.5 reagents (pair end). Libraries passing quality control were trimmed by Trim galore (v0.6.7) and mapped to the human genome hg38 using Hisat2 (v7.5.0). The reads counting was used featureCounts (v2.0.1). DESeq2 (v1.34.0) was used to compute differential gene expression using raw read‐counts as input. Heatmaps were generated using the pheatmap package (v1.0.12) in R (4.1.2).

A strand-specific library was constructed using VAHTS Total RNA-Seq (H/M/R) Library Prep Kit for Illumina following the manufacturer's instructions. Ribosome RNA was removed for retain circRNAs. The enriched circRNAs were fragmented by using fragmentation buffer and reverse transcribed into cDNA with random primers. Second strand cDNA was 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, end repaired, poly(A) 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 HiSeqTM 2500.

The target region PCR products (10 µl) were purified by adding VAHTS™ DNA Clean Beads at a 1:1 ratio, and then barcode indexing and Illumina adapters were added by Solexa PCR (20 µl reaction volumes), which used 5 µl of pooled PCR product, 2.5 µl of each index (forward and reverse), and 10 µl of 2× Q5 High-Fidelity Master Mix (NEB, USA) [19 (link)]. The PCR conditions were 98 °C for 30 s, followed by 10 cycles of 98 °C for 10 s, 65 °C for 30 s, and 72 °C for 30 s, and a final extension step of 5 min at 72 °C. Agarose gel electrophoresis was performed on a 1.8% (w/v) agarose gel, and the results were quantified by the ImageJ program. Each sample was mixed by aspirating 150 ng, and the mixed samples were purified before gel cutting using the Cycle Pure Kit and recovered by the Monarch DNA kit. The final gene library was evaluated on a Qsep400 (BiOptic, Taipei, Taiwan) for concentration and quality and then sequenced on an Illumina NovaSeq 6000. The 16S rRNA gene library construction and sequencing were completed by Biomarker Technologies Corporation (Beijing, China).

The DNA samples were used with a TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme #TD501) to build the library and VAHTS DNA Clean Beads (Vazyme #N411) for purification. The DNA libraries of 14 samples were sequenced with high-throughput sequencing technology, and each sample contained 30 G of data. The original data obtained by sequencing were converted into sequence data by base calling, which we refer to as raw data or raw reads, and the results were stored in FASTQ (Chen et al., 2018 (link)) format after filtering of low-quality data.