Long non-coding sequencing-Illumina
Service Advantages
● Service Advantages
● Cellular and tissue specific
● Specific stage expresses and presents dynamic expression change
● Precise patterns of time and space expression
● Joint analysis with mRNA data.
● 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 |
10 Gb |
Q30≥85% |
|
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 |
Nucleotides:
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.LncRNA classification
LncRNA predicted by the four softwares above were classified into 4 categories: lincRNA, anti-sense-LncRNA, intronic-LncRNA; sense-LncRNA. LncRNA classification was shown in the histogram below.
LncRNA classification
2.Cis-targeted genes of DE-lncRNA enrichment analysis
ClusterProfiler was employed in GO enrichment analysis on cis-targeted genes of differentially expressed lncRNA (DE-lncRNA), in terms of biological processes, molecular functions and cellular components. GO enrichment analysis is a process to identify DEG-directed significantly enriched GO terms compared to whole genome. The enriched terms were presented in histogram, bubble chart, etc. as shown below.
Cis-targeted genes of DE-lncRNA enrichment analysis -Bubble chart
3. By comparing the length, exon number, ORF and expression amount of mRNA and lncRNA, we can understand the differences in structure, sequence and so on between them, and also verify whether the novel lncRNA predicted by us conforms to the general characteristics.
BMK Case
Deregulated lncRNA expression profile in the mouse lung adenocarcinomas with KRAS‐G12D mutation and P53 knockout
Published: Journal of Cellular and Molecular Medicine，2019
Sequencing strategy
Illumina
Sample collection
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 investigate the aberrantly expressed lncRNAs in the mouse lung adenocarcinoma with P53 knockout and the KrasG12D mutation.
1.6424 lncRNAs were differentially expressed (≥ 2-fold change, P < 0.05).
2.Among all 210 lncRNAs(FC≥8), 11 lncRNAs’ expression was regulated by P53, 33 lncRNAs by KRAS and 13 lncRNAs by hypoxia in the primary KP cells, respectively.
3.NONMMUT015812, which was remarkably up-regulated in the mouse lung adenocarcinoma and negatively regulated by the P53 re-expression, was detected to analyse its cellular function.
4.Knockdown of NONMMUT015812 by shRNAs decreased proliferation and migration abilities of KP cells. NONMMUT015812 was a potential oncogene.
KEGG pathway analysis of the differentially expressed genes in the NONMMUT015812-knockdown KP cells |
Gene Ontology analysis of the differentially expressed genes in the NONMMUT015812-knockdown KP cells |
Reference
Deregulated lncRNA expression profile in the mouse lung adenocarcinomas with KRAS‐G12D mutation and P53 knockout[J]. Journal of Cellular and Molecular Medicine, 2019, 23(10). DOI： 10.1111/jcmm.14584
