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LncRNA Sequencing

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CD Genomics is offering a high-throughout, low cost as well as rapid turnaround lncRNA sequencing service by combining the latest Illumina sequencing instruments and advanced bioinformatics analysis.

lncRNA Sequencing

Non-protein-coding RNAs (ncRNAs) constitute a substantial portion of transcribed sequences with structural, regulatory or unknown functions. Long non-coding RNAs (lncRNAs) are defined as a large and diverse class of transcribed RNAs greater than 200 nt that do not encode for proteins, which are widely distributed in organisms and lncRNA transcripts account for the major part of the non-coding transcriptome. LncRNAs are generally classified into three groups based on their genomic regions: (i) long intergenic ncRNAs (lincRNAs), (ii) intronic ncRNAs (incRNAs) and (iii) natural antisense transcripts (NATs), which are transcribed from the complementary DNA strand of their associated genes. LncRNAs resemble mRNAs because they are typically transcribed from active chromatin, polyadenylated, and capped; however, they do not direct protein synthesis. Some differences between lncRNA and mRNA are summarized in table below.

Protein coding transcript Non-protein coding, regulatory transcript
Well conserved between species Poorly conserved between species
Present in both nucleus and cytoplasm Many predominantly nuclear, others nuclear and/or cytoplasmic
Total 20-24,000 mRNAs Currently ~30,000 lncRNA transcripts, predicted 3-100 fold of mRNA in number
Expression level: low to high Expression level: very low to moderate

LncRNAs are functionally important to organisms and not merely the product of transcriptional noise. A myriad of molecular functions have been discovered for lncRNAs in mammals and plants, including nucleosome repositioning, chromatin remodeling, transcriptional control, and posttranscriptional processing. LncRNAs are increasingly being implicated in disease occurrence, genomic imprinting and developmental regulation.

The application of next-generation sequencing technology greatly facilitated the discovery and analysis of lncRNAs. LncRNA sequence information can be acquired with single-base resolution via the high-throughput sequencing by constructing the strand-specific library for the removal of rRNA, and identify the known lncRNA and predict the new one and its target region by leveraging of the powerful bioinformatics analysis platform. The identification of long non-coding RNAs (lncRNAs) relies on the detection of transcription from genomic regions that are not annotated as protein coding, such as regions that are devoid of open reading frames. Alternatively, looking for lncRNAs and mRNAs whose expression is correlated, can perhaps indicate co-regulation or related functions.

Sequencing Workflow

To construct lncRNA sequencing library, the first step of lncRNA sequencing is to deplete rRNA, followed by RNA fragmentation, cDNA synthesis, adaptor ligation, size selection and PCR enrichment. The workflow of lncRNA sequencing process is illuminated in Figure 1.

IncRNA Sequencing Figure 1.  Schematic workflow of small RNA sequencing process.

Sequencing Strategy and Recommended Depth

  • Illumina HiSeq PE150
  • ≥ 10 Gbase for small genome and ≥ 12 Gbase for large genome

Data Analysis

Our standard bioinformatics analysis for lncRNA sequencing data includes Removal of the remaining rRNA reads by alignment/mapping to rRNA database, transcripts assembly, Identification of known transcripts and prediction of novel transcripts (including lncRNAs), quantification and differential expression analysis of lncRNAs and mRNAs, classification of lncRNA family, lncRNA functional annotation, SNP/InDel calling, identification of splicing variants, prediction of target genes and lncRNA-mRNA interaction analysis.

Sample Requirements

  1. Sample type: Total RNA without degradation or DNA contamination.
  2. Starting amount of total RNA: ≥ 2 µg
  3. Sample conc.: ≥ 200 ng/µl
  4. Sample purity: OD260/280 = 1.8~2.2

Key Features and Advantages

  • High coverage and accuracy: LncRNA sequence information of all single-base resolution can be obtained in one time.
  • Comprehensive bioinformatics analysis: we have experienced technicians, professional bioinformatics team equipped with supercomputing facilities, we use widely accepted mainstream software and mature in house pipeline.
  • High utilization: Simultaneously obtain the LncRNA and mRNA data for the joint analysis.
  • Cost-effective: Our high success rates and quality data prevent costly repeat experiment and sequencing.

Our PhD-level bioinformatics team provides comprehensive analysis for both lncRNAs and mRNAs, enabling access to lncRNA and mRNA information in a single sequencing run. We can also help in the experimental design at the very beginning of your project and offer consultation at every stage of the project process.

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