Whole transcriptome sequencing – Illumina
Service Advantages
● Estimation on all types of RNAs in terms of counts, expression and chromosome-based relative expression
● Identification of differentially expressed RNAs and corresponding expression
● Gene co-expression analysis
● ceRNA network analysis
● Key genes involved pathway analysis
● 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 |
|
Total RNA |
Illumina PE150&SE50 |
Circ/lnc/mRNA: 16G; miRNA: 10M reads |
Q30≥85% |
Sample Requirements:
Nucleotides:
| Purity | Integrity | Contamination | Amount |
| OD260/280≥1.7-2.5； OD260/230≥0.5-2.5； | For plants: RIN≥6.5; For animals: RIN≥7; 28S/18S≥1.0; limited or no baseline elevation | Limited or no protein or DNA contamination shown on gel. | Conc. ≥100 ng/μl; Volumn ≥ 10 μl; Total ≥ 2 μg |
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:
1.Dry-ice: Samples need to be packed in bags and buried in dry-ice.
2.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.Estimation on all types of RNAs based relative expression
Circos on the significancy of the differences in RNA expression
2.Integrated KEGG pathway network
Integrated KEGG pathway network
3.ceRNA network analysis
ceRNA Network-based DE-circRNA-miRNA-mRNA Interactions
BMK Case
CircRNA Expression Pattern and ceRNA and miRNA–mRNA Networks Involved in Anther Development in the CMS Line of Brassica campestris
Published: International Journal of Molecular Sciences，2019
The NONMMUT015812-knockdown KP (shRNA-2) cells and negative control (sh-Scr) cells were obtained on day 6 of a specific viral infection.
Key results
This study established the Polima cytoplasm male sterility (CMS) line “Bcpol97-05A”, and the fertile line, “Bcajh97-01B”, in Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis, and performed RNA expression profiling comparisons between the flower buds of the sterile line and fertile line by whole-transcriptome sequencing.
1.A total of 31 differentially expressed (DE) circRNAs, 47 DE miRNAs, and 4779 DE mRNAs were identified.
2.The gene ontology (GO) analysis demonstrated that Most of the DE genes were involved in pollen wall development
3.The KEGG pathway enrichment analysis of the DE mRNAs (including the up-regulated anddown-regulated mRNAs in the sterile line compared to the fertile line) showed that “starch and sucrosemetabolism”, “phenylpropanoid biosynthesis”, and “pentose and glucuronate interconversions” werethe most enriched metabolic pathways.
KEGG pathway enrichment analysis of the differentially expressed mRNAs |
Gene ontology (GO) classification of the differentially expressed mRNAs |
View of the DEcircRNA–DEmiRNA–DEmRNA triple network |
Reference
Liang Y , Zhang Y , Xu L , et al. CircRNA Expression Pattern and ceRNA and miRNA–mRNA Networks Involved in Anther Development in the CMS Line of Brassica campestris[J]. International Journal of Molecular Sciences, 2019, 20(19):4808-. DOI： 10.3390/ijms20194808
