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    Research Services

    Small RNA Sequencing

    Overview

    Novogene offers comprehensive Small RNA Sequencing service (sRNA-seq), to investigate the regulatory network of noncoding RNA of 18-40nt in length, especially for microRNA (miRNA) transcripts. Variations in miRNA can be correlated with gene silencing and post-transcriptional regulation of gene expression, which provides researchers an effective method of regulating target on mRNAs with unprecedented sensitivity and high resolution. Bioinformatic analysis of sRNA-seq illustrates differential expression of miRNAs, structural alterations and discovery of novel small RNAs via a high throughput research technique.

    Service Specifications

    Applications

  • Expression quantification of small RNA transcripts
  • Function verification, such as gene knockout, over-expression of miRNA genes
  • Advanced Analysis: miRNA target gene verification
  • Advanced Analysis: piRNA identification and expression quantification
  • Advantages

  • Extensive experience with thousands of samples being successfully sequenced.
  • Unsurpassed data quality with a guaranteed Q30 score ≥ 85% that exceeds Illumina’s official benchmarks.
  • Comprehensive analysis using mainstream software and mature in-house pipeline to meet multiple bioinformatic requests.
  • Free correlation analysis for both small RNA and mRNA expression levels to investigate the regulatory networks.
  • Sample Requirements

     

    Library Type
    Sample Type
    Amount
    RNA Integrity Number
    (Agilent 2100)
    Purity
    (NanoDrop)
    Small RNA Library
    Total RNA
    ≥ 2 μg
    Animal ≥ 7.5, Plant ≥ 7, with smooth baseline;
    OD260/280 = 1.8-2.2;
    OD260/230 ≥ 1.8;
    Exosomal Small RNA 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
    Single-end 50
    Recommended Sequencing Depth
    ≥ 10 million read pair per sample
    Standard Analysis (miRNA)
  • Data Quality Control
  • Summary of Length Distribution
  • Common and Specific Sequence Summary
  • Identification & Characterization of miRNAs
  • Classification and Annotation of miRNA
  • Quantification & Differential Expression Analysis
  • Functional Enrichment Analysis
  • Note: For detailed information, please refer to the Service Specifications and contact us for customized requests.

    Project Workflow

    Sampling:

    Small RNA library: inflorescence tissues of three-week-old transgenic seedlings in Col-0 or the ilp1-1 mutant;
    mRNA library: total RNA was isolated from 7-day-old seedlings grown under constant white light.

    Sequencing Strategy:

    1. TruSeq Small RNA Library Preparation Kit, SE50 using the Illumina Hiseq 2500
    2. strand-specific mRNA library, 2 × 150-bp reads using the Illumina Hiseq X Ten platform

    Figure 1. Summary and some examples of significantly affected alternative splice sites between ilp1-1 and ntr1-1 plants.
    Conclusion:

    This study shows that two conserved disassembly factors of the ILS complex, Increased Level of Polyploidy1-1D (ILP1) and NTC-Related protein 1(NTR1), positively regulate microRNA (miRNA) biogenesis by facilitating transcriptional elongation of MIRNA(MIR) genes in Arabidopsis thaliana(Figure1, 2). ILP1 and NTR1 forms a stable complex and co-regulates alternative splicing of more than a hundred genes across the Arabidopsis genome, including some primary transcripts of miRNAs (pri-miRNAs)(Figure3, 4). These results provide further insights into the regulatory role of spliceosomal machineries in the biogenesis of miRNAs.

    Integrated miRNA-mRNA analysis reveals regulatory pathways underlying the curly fleece trait in Chinese tan sheep

    Background:

    Tan sheep is an indigenous Chinese breed well known for its beautiful curly fleece. One prominent breed characteristic of this sheep breed is that the degree of curliness differs markedly between lambs and adults, but the molecular mechanisms regulating the shift are still not well understood. In this study, we identified 49 differentially expressed (DE) microRNAs (miRNAs) between Tan sheep at the two stages through miRNA-seq, and combined the data with that in our earlier Suppression Subtractive Hybridization cDNA (SSH) library study to elucidate the mechanisms underlying curly fleece formation.

    Sampling:

    Skin tissue was collected from the shoulder of four female Chinese Tan sheep (two 1-month-old lambs and two 48-month-old adults)

    Sequencing Strategy:

    NEBNext® Multiplex Small RNA Library Prep Set for Illumina®, Illumina HiSeq 2500/2000 platform.

    Figure 2. Analysis of differential miRNA expression between lambs and adults.
    Table 1 Differentially expressed miRNAs between lamb (L) and adult (A) Group.

    miRNA
    L_readcount
    A_readcount
    Log2 Fold Change
    P-Value
    P-Adj
    oar-miR-148a
    67, 574.96753
    231, 061.6487
    -1.7737
    1.00E-09
    0
    oar-miR-136
    19.75893919
    88.81995869
    -2.1684
    6.98E-10
    2.81E-09
    oar-miR-150
    50.50740147
    235.578046
    -2.2216
    3.78E-11
    4.18E-11
    oar-miR-29a
    867.6115945
    5480.855957
    -2.6593
    1.00E-09
    0
    novel_459
    0
    20.61202625
    -5.3654
    3.82E-06
    1.82E-05

    Figure 3. Gene Ontology (GO) analysis.
    Table 2 KEGG pathways enriched for target genes of 28 miRNAs with higher expression in skin of lambs with curly fleece.

    KEGG Pathway
    Count
    P-Value
    Corrected P-Value
    Metabolic pathways
    180
    0.894548594
    0.984724863
    Pathways in cancer
    66
    0.179111564
    0. 939563595
    PI3-Akt signaling pathway
    64
    0.39499336
    0. 939563595
    HTLV-I-Infection
    50
    0.192543289
    0.895292925
    Phagosome
    45
    0. 0017584833
    0.480065813
    Protein processing in endoplasmic reticulum
    43
    0.007929522
    0.545550536
    Regulation of actin cytoskeleton
    43
    0.245615216
    0.939563595
    MAPK signaling pathway
    42
    0.630365388
    0.939563595
    Tuberculosis
    41
    0.051840799
    0.750257819
    Ras signaling pathway
    41
    0.474634575
    0.939563595
    Viral carcinogenesis
    39
    0.270545221
    0.947474735
    Transcriptional misregulation in cancer
    37
    0.104993737
    0.779793658
    Influenza A
    37
    0.185182388
    0.939563595
    Endocytosis
    36
    0.500604613
    0.957112583
    Epstein-Barr virus infection
    35
    0.270987612
    0.947474735
    Lysosome
    33
    0.01255486
    0.638411076
    MicroRNAs in cancer
    33
    0.443228539
    0.939563595
    Focal adhesion
    33
    0.531854202
    0.957112583
    cGMP-PKG signaling pathway
    31
    0.425371479
    0.939563595
    AMPK signaling pathway
    30
    0.071269601
    0.779793658
    Conclusion:

    This study explores the role of miRNAs in the curly fleece trait of Chinese Tan sheep. This study represents the comprehensive analysis of mRNA and miRNA in Tan sheep and offers detailed insight into the development of curly fleece as well as the potential mechanisms controlling curly hair formation in humans. The results provide important clues for elucidating the molecular mechanism underlying curly fleece and curly hair development.

    Uncovering anthocyanin biosynthesis related microRNAs and their target genes by small RNA and degradome sequencing in tuberous roots of sweetpotato

    Background:

    Purple-fleshed sweetpotato (PFSP) is a desirable resource for functional food development because of the abundant anthocyanin accumulation in its tuberous roots. Some studies have shown that the expression regulation mediated by miRNA plays an important role in anthocyanin biosynthesis in plants. However, few miRNAs and their corresponding functions related to anthocyanin biosynthesis in tuberous roots of sweetpotato have been known.

    Sampling:

    Total RNA extracted from the tuberous roots of WFSP (Xushu-18) and PFSP (Xuzishu-3)

    Sequencing Strategy:

    NEBNext® Multiplex Small RNA library Prep Set for Illumina®, sequenced on an Illumina Hiseq 2000 platform

    Figure. 4 The differentially expressed miRNAs in XS-18 and XZS-3.

    Figure. 5 KEGG pathway analysis of the target genes regulated by the differentially expressed ib-miRNAs.
    Conclusion:

    The results represented a comprehensive expression profiling of miRNAs related to anthocyanin accumulation in sweetpotato and provided important clues for understanding the regulatory network of anthocyanin biosynthesis mediated by miRNA in tuberous crops. Our findings provided comprehensive information for anthocyanin-specific miRNAs and their targets, as well as a starting point for mechanism investigation of miRNAs in anthocyanin biosynthesis in sweetpotato.


    sRNA Length Distribution


    sRNA Classification-Repeat Sequence


    miRNA-structure


    miRNA-base bias


    TPM Boxplot


    TPM Density Distribution


    Hierarchical Cluster