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Long non-coding RNA Sequencing

Overview

Long non-coding RNA sequencing service (lncRNA-seq) is a comprehensive next-generation method to detect the transcripts with a length of over 200nt, which do not encode protein and perform as regulatory elements in multiple biological processes. However, the progressive library preparation enables information enrichment and gene expression profiling for both coding and non-coding transcripts from a high-sensitive transcriptomic perspective. Bioinformatic analysis does not reveals the quantification and functional enrichment of the target transcripts, but also indicates the strand orientation and regulatory relation of lncRNA targeted mRNA.

Service Specifications

Applications

  • Expression Quantification of lncRNA transcripts
  • Gene or RNA subcellular
    Localization and Expression
  • Function Verification, such as gene knockout, over-expression of lncRNA genes
  • Protein Interaction

Advantages

  • Extensive experience with numerical samples being successfully sequenced.
  • Unsurpassed data quality with a guaranteed Q30 score ≥ 80% that exceeds Illumina’s official benchmarks.
  • Comprehensive analysis using mainstream software and mature in-house pipeline to meet multiple bioinformatic requests.

Sample Requirements

Library Type Sample Type Amount RNA Integrity Number
(Agilent 2100)
Purity
(NanoDrop)
lncRNA Library Total RNA ≥ 2 μg Animal ≥ 6.5, Plant ≥ 6, with smooth baseline OD260/280 = 1.8-2.2;
OD260/230 ≥ 1.8;
Exosomal lncRNA Library Exosomal RNA ≥ 20ng Peak between 25-200nt, FU> 10, no peak > 2000nt

Sequencing Parameters and Analysis Contents

Platform Type Illumina Novaseq 6000
Read Length Paired-end 150
Recommended Data Output ≥ 40 million read pair per sample
Standard Data Analysis
  • Data Quality Control
  • Structural Analysis (Alternative Splicing(AS) & Variation Calling)
  • lncRNA Identification & Annotation
  • Expression Quantification & Differential Expression Profiling
  • Functional Enrichment Analysis
  • Protein-Protein Interaction (PPI) analysis
  • lncRNA Target Gene Prediction

Note: For detailed information, please refer to the Service Specifications and contact us for customized requests.

Project Workflow

Interferon-inducible lncRNA IRF1-AS represses esophageal squamous cell carcinoma by promoting interferon response

Background:

Interferons (IFNs) play crucial roles in the development and treatment of cancer. Long non-coding RNAs (lncRNAs) are emerging molecules involved in cancer progression. IFN-regulated lncRNAs regulate immune-related genes and may perform a function in the antiviral response. However, the roles of IFN-regulated lncRNAs in cancer and their interactions with the IFN pathway remain unclear.

Sampling:

The IFN-β-treated and control KYSE30, KYSE180 and KYSE450 cells and the IRF1-AS knockdown and control KYSE30 and KYSE180 cells were used to perform RNA sequencing (RNA-seq).

Sequencing Strategy:

RNA-seq was performed using Illumina HiSeq 4000 and was carried out by Novogene (China).

Figure 1. Identification of the IFN-regulated nuclear lncRNA IRF1-AS in ESCC.

Figure 2. Global mRNA analysis revealing that IRF1-AS regulates IFN responses. (A) RNA-seq of IRF1-AS stable-knockdown cells.
Conclusion:

In this study, the IFN regulated lncRNAs candidates (IRF1-AS, Interferon Regulatory Factor 1 Antisense RNA) were identified , which function as positive regulators of the IFN response and interferons induce IRF1-AS expression through the JAK-STAT pathway. And the IRF1-AS upregulates IRF1 in cis by forming a transcription-activating complex with ILF3 (Interleukin Enhancer Binding Factor 3) and DHX9 (DExHBox Helicase 9). Considering the regulatory network among IFN signaling, IRF1-AS and IRF1, the related molecules may be potential predictive biomarkers and targeting the feedback loop between IRF1-AS and IRF1 to enhance the IFN response may provide a novel therapeutic strategy in ESCC (Esophageal Squamous Cell Carcinoma).

Transcriptional landscape of pathogen-responsive lncRNAs in rice unveils the role of ALEX1 in jasmonate pathway and disease resistance

Background:

Plant defence is multilayered and is essential for surviving in a changing environment. The discovery of long noncoding RNAs (lncRNAs) has dramatically extended our understanding of post-transcriptional gene regulation in diverse biological processes. However, the expression profile and function of lncRNAs in disease resistance are still largely unknown, especially in monocots.

Sampling & Sequencing Strategy:

Figure 3. Workflow of experiments

Figure 4. Expression profiles of rice lncRNAs responsive to Xoo infection.

Figure 5. GO enrichment and KEGG pathway analyses of cis targets of Xoo-responsive lncRNAs.

Figure 6. Regulatory network between lncRNAs and JA-related mRNAs.
Conclusion:

In this study, target analyses of sequenced lncRNAs showed that lncRNAs associated with the accumulation of jasmonates (JA) after Xoo infection. The biosynthesis and signaling of JA pathway are activated by elevated lncRNA ALEX1 expression in rice, and ALEX1 increases endogenous JA levels, conferring broad-spectrum resistance to the bacterial pathogens. This study reveals the expression of pathogen-responsive lncRNAs in rice and provides novel insights into the connection between lncRNAs and JA pathway in the regulation of plant disease resistance.

Effects of long term antiprogestine mifepristone (RU486) exposure on sexually dimorphic lncRNA expression and gonadal masculinization in Nile tilapia (Oreochromis niloticus)

Background:

Mifepristone (RU486), a clinical abortion agent and potential endocrine disruptor, binds to progestin and glucocorticoid receptors and has multiple functional importance in reproductive physiology. A long-term exposure of RU486 resulted in masculinization of female fish, however, the epigenetic landscape remains elusive.

Sampling:

All treatments were started from 5 days after hatching (dah) in aerated fresh water and 50% water daily exchange. Fish were fed daily with commercial diet mixed with RU486 (500 μg/g, Sigma). 120 dah, two gonadal pools were prepared for RNA isolation from five control fish and fifteen RU468-treated groups.

Sequencing Strategy:

Illumina Hiseq 2000 platform and 100 bp paired-end reads were generated.

Figure 7. Candidate lncRNAs filtered by the CNCI*, PFAM** and CPC*** assemblies.*CNCI: Coding-Non-Coding-Index, PFAM**: Pfam Scan and CPC***: Coding Potential Calculator.

Figure 8. Classification of gonadal lncRNAs of tilapia.

Figure 9. The variation in differentially expressed lncRNAs under RU486 treatment.
Conclusion:

A long-term RU486 exposure induces sex reversal in genetic female fish partially through epigenetic regulation via inhibiting the expression of female specific lncRNAs, and simultaneously enhancing male specific lncRNA genes. Further investigations through functional analysis are critical to unfold the epigenetic mechanisms of lncRNA’s involvement in RU486 induced sex reversal of XX female fish by gene knockout and overexpression technology.


Alternative Splicing


lncRNA Identification


Group Comparison


Volcano Plot of Transcript Quantification


Clustering of mRNA


Clustering of lncRNA


Clustering of TUCP

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