Ribominus eukaryote kit

The total RNA was extracted from three pairs of fresh frozen TNBC and adjacent normal tissues by using TRIzol reagent (Invitrogen, CA, USA), followed by treatment with the RiboMinus Eukaryote Kit (Qiagen, Valencia, CA) to delete ribosomal RNA according to the manufacturer’s guidelines. Next, the processed RNAs were subjected to perform deep sequencing with an Illumina HiSeq 3000 (Illumina, San Diego, CA).
The RNA-seq FASTQ reads were first aligned to the human reference genome (GRCh37/hg19) by TopHat2 [22 (link)]. The sequences that aligned contiguously and full length to the genomes were discarded. Then, the remaining reads were used to identify circRNAs [14 (link)]. SRPBM (spliced reads per billion mapping) was applied to normalize the counts of reads mapping across an identified backsplice, and differential expression analysis was conducted based on the previous method [23 (link)].

Total RNA was extracted from three pairs of frozen GC tissues and paired normal tissues using TRIzol Reagent (Invitrogen, CA, USA). Ribosomal RNAs were removed using a RiboMinus Eukaryote kit (Qiagen, Valencia, CA). Then RNA was treated with RNase R (Epicentre, USA) and fragmented to ~200 bp. cDNA was synthesized with random hexamer primers and dUTPs. Uracil DNA glycosylase was used to purify cDNA. Next, the RNA sequencing (RNA-seq) library was deep sequenced with an Illumina HiSeq 3000 instrument (Illumina, San Diego, CA).

Total RNAs were extracted from three GBM and NBTs using TRIzol reagent (Life Invitrogen, Carlsbad, CA) and then treated using the RiboMinus Eukaryote Kit (Qiagen, Valencia, CA) to remove rRNAs before generating RNA-seq library. Next, the RNA-seq library was deep sequenced with the Illumina HiSeq 2000. RNA sequencing reads were aligned to the human reference genome by software STAR and RNA abundance was quantified using software RSEM.

Total RNA was extracted from normal human intestinal epithelial cell lines (HIEC-6 and NCM460) and CRC cell lines (HCT8, HCT116 and DLD1). The RNA purity was analyzed on a Bioanalyzer 2200 instrument (Aligent). Then the RNA was treated with RiboMinus Eukaryote Kit (Qiagen, Valencia, CA) to remove ribosomal RNA and a cDNA library was constructed. Finally deep sequencing was performed with an Illumina HiSeq 3000 (Illumina, San Diego, CA). The clean reads were aligned to the reference genome (GRCH37.p13 NCBI). Unmapped reads were collected to identify the circRNAs. Reads that mapped to the circRNA junction (with an overhang of at least 6 nt) were counted for each candidate.

The TRIzol reagent (Invitrogen) was employed to collect total RNA samples (3 μg), which was then treated with the RiboMinus Eukaryote Kit (Qiagen, Valencia, CA) to remove rRNA. To prepare strand-specific RNA sequencing libraries, the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Beverly, MA) was used according to the manufacturer’s instructions. In brief, random hexamer primers were used to synthesize first- and second-strand complementary DNA (cDNA) from the fragmented ribosome-depleted RNA samples (50 ng). In consideration of removal of the second strand, the second-strand cDNA was synthesized by using dUTP mix. The ends were repaired by treating cDNA fragments with the End-It DNA End Repair Kit, then cDNA fragments were modified with Klenow to add an adenosine at the 3′-end, and finally were ligated to the adaptors. The second-strand cDNA was removed from the ligated cDNA products by purifying and treating with uracil DNA glycosylase. Purified first-strand cDNA was subjected to 12–15 cycles of PCR amplification, and the libraries were quality-controlled with a Bioanalyzer 2100 (Agilent, Santa Clara, CA) and sequenced by HiSeq 2000 (Illumina, San Diego, CA, USA). The raw sequencing reads were deposited in the Gene Expression Omnibus (GEO) database under accession GSE138963.

Total RNA from 3 passages (3 biological replicates) each of MCF-7, MCF7-RES, MDA-MB-231 and MDA-RES cells was purified using the RNAiso™ Plus Kit (TaKaRa, Japan). After RNA purification and DNase I digestion, ribosomal RNAs (rRNAs) were removed from total RNA with the RiboMinus Eukaryote Kit (Qiagen, Valencia, CA). The remaining RNAs were fragmented and used to synthesize cDNAs, followed by end repairing and adenine connection. Then the sequencing adaptors were ligated to the fragments and those with suitable sizes were selected for PCR amplification. An ABI StepOnePlus System (ThermoFisher Scientific, Massachusetts, USA) and an Agilent 2100 Bioanaylzer (Agilent Technologies, California, USA) were used to quantify and qualify the sample libraries in the quality control steps. Finally, all the libraries were sequenced by an Illumina HiSeqTM 2000 sequencer.