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Long non-coding sequencing-Illumina

Long non-coding RNAs (lncRNAs) are a type of RNA molecules with length exceeding 200 nt, which are characterize by extremely low coding potential. LncRNA, as a key member in non-coding RNAs, is mainly found in nucleus and plasma. The development in sequencing technology and bioinformtics enables identification of numerous novel lncRNAs and associate those with biological functions. Accumulative evidences suggest that lncRNA is widely involved in epigenetic regulation, transcription regulation and post-transcription regulation.


Service Details

Bioinformatics

Demo Results

Case Study

Service Advantages

Ø Service Advantages
Ø Cellular and tissue specific
Ø Specific stage expresses and presents dynamic expression change
Ø Precise patterns of time and space expression
Ø Joint analysis with mRNA data.
Ø BMKCloud-based result delivery: Customized data-mining available on platform.
Ø After-sale services valid for 3 months upon project completion

Sample Requirements and Delivery

Sample Requirements:

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.
3.RNAstable tubes: RNA samples can be dried in RNA stabilization tube(e.g. RNAstable®) and shipped in room temperature.

Service Work Flow

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Experiment design

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Sample delivery

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RNA extraction

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Library construction

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Sequencing

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Data analysis

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After-sale services


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  • Bioinformatics

    CircRNA-sequencing-analysis-workflow
    CircRNA-sequencing-analysis-workflow

    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

    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 Cis-targeted genes of DE-lncRNA enrichment analysis -Bubble chart

    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.

    PB-full-length-RNA-Sequencing-case-study

    KEGG pathway analysis of the differentially expressed genes in the NONMMUT015812-knockdown KP cells

    PB-full-length-RNA-Sequencing-case-study

    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

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