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LncRNA Sequencing

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lncRNA sequencingLong non-coding RNAs (LncRNAs) are transcribed RNAs longer than 200 nt that do not encode for proteins. LncRNAs are involved in a wide range of important cellular processes such as X-chromosome inactivation, imprinting, maintenance of pluripotency, lineage commitment and apoptosis. LncRNAs are also known to be involved with human diseases such as cancer, cardiovascular disease and neurological disorders, and are of significant interest to researchers.

Novogene bioinformatics team composed entirely of PhDs provides comprehensive analysis for both lncRNAs and mRNAs, enabling access to lncRNA and mRNA information in a single sequencing run. Applications include comparison of lncRNA and mRNA expressions in different stages of development and in different tissues, as well as unveiling key functions of mRNAs and lncRNAs.

The Novogene Advantage

  • Extensive experience with thousands of samples successfully sequenced.
  • Unsurpassed data quality with a guaranteed Q30 score ≥ 80% that exceeds Illumina’s official guarantee.
  • Comprehensive analysis using widely accepted mainstream software and mature in house pipeline.
  • Free, powerful Novofinder software that enables Novogene customers to easily access and visualize data analysis results and annotations through a user-friendly interface.

Project Workflow

LncRNA Sequencing Project Workflow

Sequencing Strategy

  • 250~300 bp insert strand specific library with rRNA removal (Ribo-ZeroTM Magnetic Kit)
  • HiSeq platform, paired-end 150 bp

Data Quality Guarantee

  • Our data quality guarantee, as measured by the percentage of bases with a sequencing quality score above Q30 (PE150, ≥ 80%), exceeds Illumina’s official guarantee (PE150, ≥ 75%). See examples of our high quality data.

Sample Requirements

  • Total RNA amount: ≥ 2.0 μg; RNA concentration: ≥ 50 ng /μl
  • RIN value ≥ 6.3 for plants and fungi; RIN value ≥ 6.8 for animals
  • RNA volume: ≥ 20 μl
  • Purity: No degradation, no DNA contamination

Turnaround Time

  • Within 15 working days from verification of sample quality (without data analysis)
  • The data analysis turnaround is project-dependent.

Recommended Sequencing Depth

  • ≥ 10 Gb data

Analysis Pipeline

lncRNA Sequencing Analysis Pipeline

Table. Representative data quality results of lncRNA sequencing (PE 150) from Novogene
Sample Name# Of Raw reads# Of Clean readsClean basesError Rate (%)Q 20 (%)Q 30 (%)GC Content (%)
Sample 111872676611742406817.61G0.0197.7094.0952.33
Sample 213394847413248170219.87G0.0197.8594.4050.66
Sample 314428543014269617621.4G0.0197.8494.4252.15
Sample 413306691413208679419.81G0.0198.2795.5049.99
Sample 513357027813180171819.77G0.0198.2895.4953.05
Sample 611140069810773277616.16G0.0197.8294.3151.56

Project Example

The following studies utilized Novogene's expert lncRNA sequencing service.

Identification and functional characterization of hypoxia-Induced endoplasmic reticulum stress regulating lncRNA (HypERlnc) in pericytes
Circulation Research DOI: 10.1161/CIRCRESAHA.116.310531 (2017)

LncRNAs (long non-coding RNAs) have significant regulatory roles in vessel stability in pericytes. However, their function in pericyte biology is largely unknown. Novogene’s lncRNA sequencing service was adopted in this study and the Hypoxia-Induced Endoplasmic Reticulum Stress Regulating lncRNA (HypERlnc) was identified and characterized. HypERlnc knockdown showed pericyte de-differentiation and loss of pericyte function, as well as induced ER stress. RNA seq and subsequent Gene Ontology (GO) and KEGG analysis revealed the role of HypERlnc in human cardiopulmonary disease. This study demonstrates that lncRNA sequencing, combined with other approaches such as RT-qPCR, can reveal previously unexplored and complex characteristics and functions of HypERlnc.



Figure. RNA-seq in primary mouse pericytes demonstrates high read coverage of the possible mHypERlnc orthologue (in purple)


Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells
Nature Structural & Molecular Biology 20, 1131–1139 (2013)

Measuring gene expression in individual cells is crucial for understanding the gene regulatory network controlling human embryonic development. Here we apply single-cell RNA sequencing analysis to 124 individual cells from human preimplantation embryos and human embryonic stem cells (hESCs) at different passages. The number of maternally expressed genes detected in our data set is 22,687, including 8,701 long noncoding RNAs (lncRNAs), which represents a significant increase from 9,735 maternal genes detected previously by cDNA microarray. We discovered 2,733 novel lncRNAs, many of which are expressed in specific developmental stages. To address the long-standing question whether gene expression signatures of human epiblast (EPI) and in vitro hESCs are the same, we found that EPI cells and primary hESC outgrowth have dramatically different transcriptomes, with 1,498 genes showing differential expression between them. This work provides a comprehensive framework of the transcriptome landscapes of human early embryos and hESCs.

lncrna-data-graphs
Figure.
Expression patterns of known long noncoding RNA (lncRNA) genes during human preimplantation development and derivation of hESCs.


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