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)].
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)].
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