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Sample GSM3346381

Query DataSets for GSM3346381

Status

Public on Dec 31, 2018

Title

CCML_sequence

Sample type

SRA

Source name

TSCC tissue specimens

Organism

Homo sapiens

Characteristics

tissue: tongue
tissue type: Tongue squamous cell carcinoma (TSCC) tissue specimens
molecule subtype: circular RNA

Extracted molecule

total RNA

Extraction protocol

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Basecalls performed using CASAVA version 1.8
circRNA-seq reads were mapped to human genome by BWA-MEM software by default parameters except -T = 19
circRNAs were detected and annotated with CIRI software
Genome_build: hg19
Supplementary_files_format_and_content: Number of raw junction read

Submission date

Aug 19, 2018

Last update date

Dec 31, 2018

Contact name

hao wu

E-mail(s)

[email protected]

Phone

13706294560

Organization name

Affiated hospital of nantong unversity