Fresh TSSC tissue specimens (n=3) and adjacent tissue specimens (n=3) were homogenized. Total RNA was extracted from the frozen TSCC tissues and adjacent tissues with using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). The RNA concentration of each sample was measured by NanoDrop ND-1000 analysis (Agilent Inc. USA). The OD260/OD280 ratio was used as the RNA purity index. The RNA purity is qualified if the OD260/OD280 ratio ranges from 1.8 to 2.1, then QC Results are marked as "Pass". Total RNA from each sample was used to prepare the circRNA sequencing library, which included the following steps:1) 5 μg total RNA were pretreated to enrich circRNA using CircRNA Enrichment Kit (Cloud-seq Inc, USA). 2) RNA libraries were constructed by using pretreated RNAs with TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions.3) Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). The libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as clusters and finally sequenced for 150 cycles on Illumina HiSeq Sequencer according to the manufacturer's instructions.Paired-end reads were harvested from Illumina HiSeq Sequencer after quality filtering. The reads were aligned to the reference genome/transcriptome with BWA-MEM software and circRNAs were detected and annotated with CIRI software. circBase database and circ2Trait disease database were used to annotated the identified circRNAs. Raw junction reads for all samples were normalized by total reads number and log2 transformed. Differentially expressed circRNAs were identified by t-test between two groups. Fold change >2.0 and P-value ≤0.05 were used as cutoffs for differential circRNAs. R package was used to perform cluster analysis for the normalized junction reads that were differentially expressed.
GEO Accession
GSM3346382