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Small RNA Sequencing

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.

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

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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

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Note: Sequencing depths and bioinformatic analysis(or advanced analysis for mRNA association or piRNA) requests can be customized based on the project needs. Please contact us for more information.

Project Workflow

Sample Quality Control

Library Quality Control

Data Quality Control

Sample Preparation

Library Preparation

Sequencing

Bioinformatics Analysis

Spliceosome disassembly factors ILP1 and NTR1 promote miRNA biogenesis in Arabidopsis thaliana

Background:

MiRNAs are a class of endogenous small non-coding RNAs that predominantly mediate post-transcriptional gene silencing (PTGS). The intron-lariat spliceosome (ILS) complex is highly conserved among eukaryotes, and its disassembly marks the end of a canonical splicing cycle. But the regulation of miRNA biogenesis by spliceosome-associated proteins and the plant ILS complex remains limited and ambiguous.

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.

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

 

Table 1 Differentially expressed miRNAs between lamb (L) and adult (A) Group.

Figure 3. Gene Ontology (GO) analysis.

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

 

Table 2 KEGG pathways enriched for target genes of 28 miRNAs with higher expression in skin of lambs with curly fleece.
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