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Transcriptomics Q&A

  • General Questions

  • Which kinds of transcriptome sequencing can be chosen?
    • (1) According to the type of RNA
      There are mRNA sequencing, SmallRNA sequencing, LncRNA sequencing, CircRNA sequencing, whole transcriptome sequencing, etc.
      (2) According to species characteristics<
      For example, eukaryotes or prokaryotes, whether there is a reference genome, different sequencing platforms, eukaryotic transcriptome sequencing with and without reference, prokaryotic transcriptome sequencing, full-length transcriptome sequencing, etc.
  • How to choose the data volume size for transcriptome sequencing?
    • The amount of data required for transcriptional regulatory sequencing varies depending on the type of project, and the amount of data is also related to genome size and complexity.
      To ensure the reliability and accuracy of data analysis results, for Illumina platform and PacBio platform: 6Gb data volume is recommended for eukaryotic transcriptome sequencing for subsequent analysis, and 8-10Gb data volume is recommended.
      If you want to detect transcripts with lower abundance; for prokaryotic transcriptome, 4Gb data volume is recommended for subsequent analysis; and for some genes with a high number of species, items of concern with lower gene expression abundance, and pathogen-host interactions, the data volume can be increased appropriately according to the situation.
  • What is the difference between prokaryotic transcriptome and eukaryotic transcriptome libraries?
    • Eukaryotic mRNA has a poly A tail and can be enriched by poly dT. In addition, eukaryotic genomes are usually larger and usually only have genes on the positive strand, so eukaryotic library building can be done by poly A enrichment.
      Prokaryotic genomes have a higher utilization rate and more genes exist in both positive and negative strands, so we need to distinguish between positive and negative strand information in prokaryotic library building, and our prokaryotic library building method is ribosomal strand-specific library building.
  • mRNA transcriptome routine library building process?
    • 1. Extraction of total RNA from the sample
      2. Oligo(dT) magnetic beads enrichment of RNA with ploy A structure at the 3' end
      3. Reverse transcription and construction of cDNA library
      4. PCR
      5. Double-end PE250 sequencing based on Illumina NoveSeq 6000 platform
  • What is the relationship between transcriptome sequencing and DEG (digital gene expression)?
    • The main differences between transcriptome and DEG are sequencing strategy, data volume, and data analysis. Transcriptome sequencing has a larger data volume and allows for gene structure analysis in addition to analysis of differentially expressed genes. In contrast, DEG sequencing data volume is small, which generally means that it can satisfy differential gene expression analysis and cannot perform gene structure analysis.
  • Does transcriptome sequencing require biological replicates?
    • Individual organisms are very different, and if the sample size is small, the differentially expressed genes obtained are likely to be the expression of only a few individual differences, and do not reflect the essential characteristics of a disease or a particular physiological state of the population.
      For plant and animal samples, more than 5 biological replicates are recommended to do correlation tests between biological samples to improve the credibility of the experimental results.
      For cell samples, the variability between biological replicates is relatively small, and more than 3 biological replicates are recommended.
      For samples of clinical studies, more biological replicates are required due to possible differences in the genotype, lifestyle, living environment, age and gender of the donors, and more than 10 biological replicates are generally required.
  • When sequencing the eukaryotic transcriptome, will mitochondrial and chloroplast RNA be detected?
    • Normally not, because mitochondrial and chloroplast RNAs do not have poly-A tails.
  • Can I do a prokaryotic transcriptome or reference-free transcriptome analysis when the species does not have a reference genome?
    • No, it is not recommended. Prokaryotic transcriptome requires a reference genome because mRNAs of prokaryotes are generally multiple cis-trans, and direct splicing is less effective and has a great impact on subsequent data analysis.
  • For the case of bad reference genome, should I choose to sequence the transcriptome without or with reference?
    • As long as the reference genome is available, it is preferably recommended to use the reference genome for the analysis. The more difficult part for genome splicing is the repeat region, and the poor quality of gene assembly is mainly due to the repeat sequences. The sequences of the coding regions of the genome are relatively easy to splice because they have good heterozygosity or repeatability. The assembly quality of the coding region is generally better. Therefore, the reference genome is used preferentially.
  • What are the factors affecting transcriptome sequencing results?
    • The degradation of RNA seriously affects the quality of sequencing. After RNA degradation, the purified mRNA cannot be captured after adding poly-A, therefore, random primer reverse transcription cannot get all cDNAs, resulting in obvious 3'- and 5'-bias of sequencing results. The presence of poly-A polymorphs in the library can interfere with the sequencing signal and affect the accuracy of the sequencing results; meanwhile, due to the inconsistent abundance of transcripts in the transcriptome, the samples need to be homogenized before the experiment, otherwise the high-abundance expressed genes will mask the low-abundance expressed genes, resulting in the failure of finding new genes or obtaining a large number of meaningless duplicate sequences.
  • Why do I need to build a strand-specific library?
    • Many genes have positive- and negative-sense RNA transcripts on the genome, and antisense transcription is a feature of eukaryotic genes and is an important mode of regulation. The use of strand-specific libraries preserves the directional information of RNA and allows us to determine whether the transcripts are from positive or negative strands, allowing us to obtain more accurate gene characterization results and gene expression information.

  • Sample Preparation

  • Do I need to snap freeze my cell sample after adding trizol to RNA extraction?
    • Trizol is a reagent for extracting total RNA directly from cells or tissues. Trizol maintains the integrity of the RNA during sample lysis or homogenization; to ensure the integrity of the RNA, it needs to be snap-frozen in liquid nitrogen and then transferred to -80°C for freezing (for long-term storage).
  • Is 20 mg of adipose tissue enough for transcriptome?
    • Adipose tissue is a tissue sample with low nucleic acid content and low RNA yield. Under normal circumstances, 1 mg of adipose tissue can only extract 0-0.05 μg of RNA, 20mg of sample can only mention 1 μg of RNA at most if the sample is in good condition and without considering the extraction loss, while normally 2 μg of RNA is needed for transcriptome sequencing, it is recommended to send more samples, preferably above 50 mg.
  • What are the precautions to take during the delivery of blood samples for transcriptome sequencing?
    • It is recommended to use EDTA anticoagulation tubes for whole blood samples, or sodium citrate anticoagulation tubes are also acceptable. Blood cells can also be centrifuged, but both whole blood and blood cells must be treated with Trizol. After snap-freezing in liquid nitrogen, store at -80°C and transport on dry ice.
      For human whole blood, it is recommended to use PAXgene tubes for collection, which can be stored for 3 days at 18-25°C, 5 days at 2-8°C, and 8 years at -20 to -80°C. In principle, -80°C storage and dry ice transport are required.
  • What do I need to pay attention to during the collection of fresh tissue samples?
    • After fresh tissue samples are isolated, endogenous RNA enzymes in the samples will rapidly degrade RNA in a short period of time, and apoptosis will also affect the expression of transcript levels in fresh tissue samples. Therefore, it is recommended that fresh tissue samples be cryogenically and rapidly obtained, immediately and adequately snap frozen in liquid nitrogen, stored at -80°C and transported on dry ice. Minimize the exposure time of tissue samples to room temperature air to ensure the integrity of RNA.
  • Can tissue samples be directly stored on dry ice for transcriptome sequencing and will such storage affect the sequencing results?
    • After the tissue samples are taken down, they should be stored in RNA protection solution and then transferred to dry ice to minimize the degradation of RNA that can affect the results of subsequent sequencing. (Remark: Tissue samples should be cut and shredded to ensure that the RNA protection solution fully infiltrates the samples).
  • What should I pay attention to when preparing clinical samples such as tumor samples?
    • Tumor samples are highly active and the RNA will be almost completely degraded in a short time under room temperature conditions. Therefore, it is recommended that tumor samples be obtained at low temperature and quickly, immediately and adequately snap frozen in liquid nitrogen, stored at -80°C and transported on dry ice. Minimize the exposure time of tissue samples in room temperature air to ensure the integrity of RNA (for specific delivery requirements, please refer to our transcriptome delivery guidelines).
  • Can I use trypsin to digest walled cells for RNA-seq and collect samples?
    • 1. Remove the adherent-grown cells from the incubator, observe the cells under the microscope to determine the good growth state and discard the medium.
      2. Add 1×PBS prepared with the same volume of enzyme-free water as the medium, wash the cells for 1 min, then discard the PBS and repeat once.
      3. Add the appropriate amount of lysis solution repeatedly blowing until fully lysed (Trizol, for example, 10-15 cm dish, each time adding 1-2 mL, blowing and mixing, the criteria for adequate lysis is that the liquid is not viscous when blowing, good fluidity. If the liquid is too viscous, it means that the lysis is not sufficient and the lysis solution should be added.)
      4. After transferring to a threaded mouth lyophilization tube resistant to -192°C, -80°C refrigerator for long-term storage, dry ice transport.
      (Note: Try not to use trypsin digestion, trypsin is more test operating experience, poor operation will make the cells rupture)
  • Specimen for mRNA, the sample has been precipitated in isopropanol, can this be sent directly, or should I lift the RNA and then send the sample?
    • After adding chloroform and centrifuging, the sample is divided into an aqueous layer and an organic layer. After collecting the aqueous layer, the RNA can be reduced by isopropanol precipitation. Since the client has already done the isopropanol precipitation step, it is better to extract the RNA and then send the sample to save the extraction time.
  • Can I do general transcriptome with paraffin section samples?
    • No. Paraffin sections cannot be used for general transcriptome. Because paraffin section sample itself is very easy to degrade, the mRNA fragment in the sample may be incomplete.
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