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Original link：Genomics - lncRNA sequencing
brief introduction
Long non coding RNA (lncRNA) is a type of non coding RNA (ncRNA) with a length greater than 200 nt, which is widely present in various organisms and plays an important role in epigenetic regulation, cell cycle regulation, and cell differentiation regulation. It is closely related to the growth and development of animals and plants, as well as the occurrence of human diseases. It can also serve as a biomarker or important target for disease diagnosis.
The use of high-throughput sequencing technology for lncRNA sequencing and bioinformatics analysis can quickly and accurately identify lncRNAs with important regulatory functions, analyze their relationship with specific biological processes, and deeply explore the functions and expression regulation mechanisms of lncRNAs. This includes the ceRNA regulatory mechanism of competitive binding to miRNA, as well as a series of controls on chromatin structure such as enhancers, promoters, insulators, etc.
Sample starting quantity and sample delivery suggestions
| Sample type | initiation mass |
| Animal and clinical organ tissues/brain tissues, etc | >20mg |
| Animal and clinical skin/bone/blood vessels/adipose tissue, etc | >100mg |
| Plant leaf tissue/flowers | >200mg |
| Plant roots/stems/fruits/seeds | >500mg |
| Primary cells/cell lines | >5×106个 |
| Neutrophils/Eosinophils/Basophils | >5×107个 |
| Extracellular vesicle samples | >1×108个 |
| Serum/plasma/cerebrospinal fluid/joint effusion/follicular fluid | >2mL |
| Cell culture supernatant | >20mL |
| urine | >30mL |
| Total RNA | >1μgAndRIN>7.0 |
matters needing attention:
① It is recommended to store the tissue samples in RNAlater, RNAHold, RNAProtect and other relevant tissue preservation solutions, and then store them at -80 ℃ or send them on dry ice;
② After the cell samples are thoroughly lysed using TRIzol or other lysis buffer, they should be stored at -80 ℃ or sent on dry ice
③ For a more detailed sample preparation guide, please contact our online customer service
Bioinformatics analysis process and analysis content
| lncRNA | Analysis content | remarks |
| Sequencing data quality control | Statistics of sequencing raw data volume and clean data volume | |
| Sequencing quality Q20 Q30 GC content statistics, removing adapters, low-quality, and contaminated sequences from the original offline data | ||
| genomic alignment | Reference genome alignment/region alignment | |
| Chromosome density distribution | ||
| Expression analysis | Gene/transcript expression profile | |
| Statistical distribution of gene expression values in samples | ||
| Statistical analysis of interval distribution of gene expression values in different samples | ||
| Depth statistics of sample gene transcript coverage | ||
| Gene expression density map | ||
| Gene expression level boxplot | ||
| Sample correlation analysis | Pearson/Spermancorrelation analysis | |
| PCA clustering analysis | ||
| MRNA analysis | Gene difference analysis | |
| Transcriptome differential analysis | ||
| Differential gene volcano map | ||
| Overall statistical chart of differentially expressed genes | ||
| Differential gene clustering heatmap | ||
| Differential Gene GO Enrichment Analysis | ||
| KEGG enrichment analysis of differentially expressed genes | ||
| GSEA analysis | ||
| LncRNA analysis | Transcript reconstruction and lncRNA prediction | |
| Analysis of lncRNA expression | ||
| Differential expression analysis of lncRNA | ||
| Differential lncRNA volcano map | ||
| Overall statistics of differentially expressed lncRNAs | ||
| Differential lncRNA clustering heatmap | ||
| Comparison of structural characteristics between lncRNA and mRNA | Comparison of lncRNA and mRNA Length | |
| ORF distribution of lncRNA and mRNA | ||
| Statistics of lncRNA and mRNA exon numbers | ||
| Statistical analysis of lncRNA and mRNA expression levels | ||
| Prediction of lncRNA target genes | ||
| GO enrichment analysis of lncRNA targeting differentially expressed genes | ||
| KEGG enrichment analysis of lncRNA targeting differentially expressed genes | ||
| SNP/INDELanalysis | SNP/INDELNumber statistics | |
| Location of SNP/NDEL | ||
| SNP mutation pattern statistics | ||
| Gene region SNP and INDEL annotation | ||
| Variable Shear Analysis | Variable cutting type statistics | |
| Variable cutting visualization |
Application scenarios and cases
Application scenario 1: Promoter/enhancer regulation
Scope of application: Research on nonlinear transcriptional regulation, discovery and functional identification of novel cis acting elements, screening and determination of tissue-specific promoters, etc
The self transcription of lncRNA can interfere with the transcription of genes encoding proteins in its vicinity. When the upstream lncRNA is transcribed, it crosses the promoter region of the adjacent target gene, interfering with the binding of transcription factors to the promoter of the target gene, thereby inhibiting the transcription of the target gene. In addition, lncRNAs have the function of promoting enhancer circularization and activating gene expression. Without cyclization, enhancers are in an inactive state.
Application scenario 2: Combined with protein modification/DNA methylation/m6A
Scope of application: Clinical medicine, basic medicine, plant genetics and breeding research, etc
LncRNAs are involved in epigenetic regulation, possibly by recruiting chromatin remodeling complexes to mediate gene silencing, particularly in regulating histone methyltransferases associated with histone modifications. In addition, lncRNA can also bind to some DNA methylation and demethylase enzymes, causing methylation changes in the promoter region of genes and affecting gene expression.
Application scenario 3: ceRNA regulatory mechanism
Scope of application: Research on environmental stress, clinical medicine, plant genetics and breeding, early diagnosis of diseases, etc
The full name of ceRNA is competitive endogenous RNA, which is a functional element that can compete to bind to RNA. Usually, lncRNA and circRNA compete for binding to miRNA, and we generally refer to lncRNA and circRNA as ceRNA. The full name of the ceRNA regulatory network is ceRNA regulatory network, which refers to the entire cascade of regulatory networks involving ceRNA. And ceRNA analysis refers to the analysis of the entire ceRNA regulatory network. Generally, there are circRNA miRNA mRNA analysis or lncRNA miRNA mRNA analysis.
Project Process
Eukaryotic transcriptome sequencing can provide researchers with a complete service process from sample extraction, library sequencing, data analysis, etc., providing high-quality data results and strong reference for subsequent research.
