Sample Submission Guidelines
LncRNA Sequencing Q&A
General Questions
- What is your library building and sequencing strategy for LncRNA transcriptome sequencing?
- Extract the total RNA of the sample, remove ribosomal RNA and construct a strand-specific library, i.e. lncRNA library. Based on Illumina NoveSeq 6000 platform, double-end PE250 sequencing is performed.
Sequencing: Since the lncRNA library contains information of mRNA and lncRNA, and the abundance of lncRNA is low, the recommended data volume is 10-12G raw data/sample for mammals such as human, rat and mouse.
- Extract the total RNA of the sample, remove ribosomal RNA and construct a strand-specific library, i.e. lncRNA library. Based on Illumina NoveSeq 6000 platform, double-end PE250 sequencing is performed.
- Why should rRNA be removed when lncRNA sequencing and library construction?
- Only the 3' end of lncRNA carries polyA structure, other lncRNAs do not have polyA structure, so oligo(dT) magnetic bead enrichment is not recommended to get more complete lncRNA information.
- What are the species requirements for lncRNA sequencing?
- • The species has a reference genome, spliced at least to the scaffold level.
• A more complete annotation is available.
- • The species has a reference genome, spliced at least to the scaffold level.
- How to predict lncRNA target genes?
- LncRNA can regulate target genes by co-location and co-expression as a regulatory RNA. co-location target gene prediction is based on the principle that the function of lncRNA is related to the protein-coding genes near its coordinates, so that the protein-coding genes in the close position of lncRNA are screened out as its target genes. The co-expression target gene prediction principle suggests that the function of lncRNA is not related to the location of the coding gene, but to its co-expressed protein-coding gene. The target genes can be predicted by correlation analysis or co-expression analysis between the expression of lncRNAs and protein-coding genes between samples.
- How to explain that the predicted partial lncRNA sequences are consistent with mRNA sequences?
- • The lncRNA of sense was not removed from the database, the lncRNA of sense type is overlap with mRNA to some extent.
• If the lncRNA and mRNA positions are exactly the same, we need to consider whether they are located in the positive and negative strands respectively. LncRNA and mRNA may exist in different strands, but the bases are completely complementary.
- • The lncRNA of sense was not removed from the database, the lncRNA of sense type is overlap with mRNA to some extent.
- How to screen lncRNA when analyzing sequencing data?
- 1. Merge transcripts obtained by splicing all samples using cuffcompare software and select transcripts that are identified by both splicing software, or appear in at least two samples at the same time.
2. Select transcripts ≥ 200bp.
3. Screen transcripts by their FPKM values.
4. If known lncRNA data exist for the species, the transcripts obtained in the previous step are first compared with the known lncRNAs by cuffcompare to obtain transcripts that are identical to the known lncRNAs. This fraction of transcripts was directly incorporated into the final lncRNA set without further screening. After that, those transcripts that are similar or identical to the above known transcripts are screened out by comparing with transcripts of known non-lncRNA and non-mRNA types (rRNA, tRNA, snRNA, snoRNA, pre-miRNA, pseudogenes, etc.) of the species. If no known lncRNA data are available, then direct comparison with known non-lncRNA and non-mRNA type transcripts of the species is performed.
5. Screen candidate lincRNA, intronic lncRNA, anti-sense lncRNA and other types for subsequent quantitative analysis by comparing with known mRNA and using the information in the cuffcompare analysis results.
- 1. Merge transcripts obtained by splicing all samples using cuffcompare software and select transcripts that are identified by both splicing software, or appear in at least two samples at the same time.
- What validation experiments can be done after lncRNA sequencing?
- • Expression verification: verify the expression level of differential genes by fluorescence quantification, whether it is consistent with sequencing results.
• In situ hybridization (FISH)
• Loss of function experiment: silence lncRNA using siRNA, shRNA, antisense nucleic acid and other methods to observe the effect of intervention on cellular value-added, apoptosis, invasion, metastasis, chromosomes, etc., and verify the expression of lncRNA target genes.
• Functional acquisition experiment: construct lncRNA overexpression vector, observe the effect on cell proliferation, apoptosis, invasion, etc. after overexpression of lncRNA, and verify the expression abundance of lncRNA target genes.
• Overexpression or knockdown experiments: if lncRNA is localized to the nucleus and the expression level of predicted target genes is found to be elevated after deletion, it is speculated that lncRNA may bind to transcription factors (proteins) to inhibit DNA transcription; or lncRNA is localized in the cytoplasm and overexpression or deletion experiments find that some protein activities are elevated or reduced, it is speculated that lncRNA may bind to some proteins The lncRNAs may bind to some proteins and exert their effects.
- • Expression verification: verify the expression level of differential genes by fluorescence quantification, whether it is consistent with sequencing results.
For research purposes only, not intended for clinical diagnosis, treatment, or individual health assessments.
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