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Protocol publication

[…] cceptable. RNA integrity and DNA contamination were then assessed using electrophoresis on a denaturing agarose gel. In order to enrich pure circRNAs, linear RNAs were removed from the total RNA in each sample after treatment with RNase R (Epicentre, Inc.). The enriched circRNAs in each sample were amplified and fragmented into small pieces using divalent cations under an elevated temperature. RNA-seq library preparation was carried out using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina. Sequencing of the libraries was conducted on an Illumina HiSeq™ 3000 system following the vendor’s recommended protocol (TruSeqR RNA Sample Preparation v2 Guide, Illumina Company Ltd)., HTSeq software was used to count the reads numbers mapped to each circRNA. RPM (Reads Per Million mapped reads, including TopHat mapping and TopHat-Fusion mapping) [] was employed to calculate the expression level of individual circRNA. Differential expression between circRNAs was assessed by DEGseq algorithm. We defined the statistical criteria for selecting aberrant-expressed circRNA using a q-value < 0.05 with a fold change > 2.0 or < 0.5., Following RNA extraction from the LSCC and matching normal tissues (n = 10 pairs), cDNA was synthesized with a reverse transcriptase according to the kit’s instructions (SuperScript First-Strand Synthesis System for RT-PCR, Invitrogen, Carlsbad, CA, USA). circRNA expression was measured using qPCR (SYBR Green PCR Master Mix, Applied Biosystems, Foster City, CA, USA), conducted in a 20-μl reaction volume consisting of the following agents: 6.8 μl cDNA, 10 μl 2 x SYBR Green mix, 0.8 μl primer forward (5 μM), 0.8 μl primer reverse (5  […]

Pipeline specifications

Software tools HTSeq, TopHat, TopHat-Fusion, DEGseq
Organisms Homo sapiens