circRNA sequencing-Illumina
Service Advantages
● CircRNA is highly correlated with mRNA expression regulating
● Participated in the regulatory system of circRNA - miRNA - mRNA
● Identification and statistics on known circRNA and novel circRNA prediction
● BMKCloud-based result delivery: Customized data-mining available on platform.
● After-sale services valid for 3 months upon project completion
Sample Requirements and Delivery
|
Library |
Platform |
Recommended data |
Data QC |
|
rRNA depletion |
Illumina PE150 |
16 Gb |
Q30≥85% |
Nucleotides:
|
Conc.(ng/μl) |
Amount (μg) |
Purity |
Integrity |
|
≥ 100 |
≥ 0.5 |
OD260/280=1.7-2.5 OD260/230=0.5-2.5 Limited or no protein or DNA contamination shown on gel. |
For plants: RIN≥6.5; For animals: RIN≥7.0; 5.0≥28S/18S≥1.0; limited or no baseline elevation |
Sample Requirements:
Tissue: Weight(dry): ≥1 g
*For tissue smaller than 5 mg, we recommend to send flash frozen(in liquid nitrogen) tissue sample.
Cell suspension: Cell count = 3×107
*We recommend to ship frozen cell lysate. In case that cell counts smaller than 5×105, flash frozen in liquid nitrogen is recommended.
Blood samples:
PA×geneBloodRNATube;
6mLTRIzol and 2mL blood(TRIzol:Blood=3:1)
Recommended Sample Delivery
Container: 2 ml centrifuge tube (Tin foil is not recommended)
Sample labeling: Group+replicate e.g. A1, A2, A3; B1, B2, B3... ...
Shipment:
Dry-ice: Samples need to be packed in bags and buried in dry-ice.
RNAstable tubes: RNA samples can be dried in RNA stabilization tube(e.g. RNAstable®) and shipped in room temperature.
Service Work Flow
Experiment design
Sample delivery
RNA extraction
Library construction
Sequencing
Data analysis
After-sale services
Bioinformatics
1.CircRNA Hosts
It has been reported that majority of circRNAs are originated from exons rather than introns or intergenic regions. Moreover distribution of circRNA varies significantly among chromosomes. Therefore, it is of great value to evaluate different distributions of circRNA hosts between different samples, including distribution of that on different gene regions and on different chromosomes.
Distribution of circRNA host
2.Clustering Analysis on DE-circRNA
Hierarchical clustering of differentially expressed circRNAs
3.GO Annotation on DE-circRNA Host Genes
GO classification of DE-circRNA host genes
BMK Case
CPSF4 regulates circRNA formation and microRNA mediated gene silencing in hepatocellular carcinoma
Published: Oncogene,2021
Key results
1.CPSF4 expression is upregulated in HCC and that high expression of CPSF4 is associated with poor prognosis in HCC patients
2. CPSF4 reduces the levels of circRNAs, which possess a polyadenylation signal sequence and this decrease in circRNAs reduces the accumulation of miRNA and disrupts the miRNA-mediated gene silencing in HCC
3.Experiments in cell culture and xenograft mouse models showed that CPSF4 promotes the proliferation of HCC cells and enhances tumorigenicity. Moreover, CPSF4 antagonizes the tumor suppressor effect of its downstream circRNA in HCC
miRNA levels and protein levels |
Wang X , Dong J , Li X , et al. CPSF4 regulates circRNA formation and microRNA mediated gene silencing in hepatocellular carcinoma[J]. Oncogene.DOI： 10.1038/s41388-021-01867-6
