RNA sequencing

RNA-seq using ribosomal depletion is a good choice when you are interested in expression in bacterial samples (single isolates or mixed communities) and/or want to capture total RNA (not just mRNA). We use the Illumina Stranded Total RNA Prep with Ribo-Zero Plus. This method is necessary for bacterial species as they do not have poly adenylated RNA, so mRNA enrichment won’t work. Depletion of ribosomal RNA is critical, as rRNA can comprise as much as 90-98% of a total RNA sample. This kit requires RNA inputs, 25–500 ng purified total RNA input from high or standard-quality RNA samples (RIN >5).   

For standard RNA-sequencing for bacterial samples, we recommend 10 million read pairs (3Gb) per sample for small genomes, Our Stranded Total RNA sequencing pricing includes sequencing at an average of ~10 million read pairs per sample.  

Note: both the mRNA and Total RNA methods we use capture the stranded-ness or transcript polarity of the RNA, which is important for correct annotation of genes. Since there are many genomic regions that generate transcripts from both strands, identifying the polarity of a given transcript provides essential information about the possible function of a gene. Strand-specific RNA-Seq, also known as ‘stranded’ or ‘directional’ RNA-Seq, preserves this information during library preparation, allowing researchers to determine the orientation of the gene on the DNA template. 

Sample requirements for RNA sequencing using ribodepletion

Quality

• mid to high-quality RNA samples (RIN ≥ 5)
• RNA must be free of DNA, please include a Dnase treatment with the RNA isolation method.
• Library performance can vary with lower input amounts and lower quality RNA.
• FFPE samples are not recommended for this kit

Quantity

• Required volume: 30ul, more is better 
• Minimum concentration: 5 ng/ul
• Preferred concentration: at least 20ng/ul

Turnaround time and data output

• • Typical turnaround time is about 6 weeks.
  • .fastq data will be returned to you via cloud link. There are no additional bioinformatics offered at this time for RNA-seq. We recommend working with a skilled bioinformation to analyze your data. Additionally, Genomics Aotearoa offers free RNA seq analysis workshops as well as an online tutorial

 Small RNA sequencing enables analysis of miRNA and other small RNA species. Short RNA transcripts, such as microRNA (miRNA) and small interfering RNA (siRNA), can play important gene regulatory functions in the cell. In small RNA-Seq, RNA species are selected by size fractionation from total RNA. We use the NEXTFLEX® Small RNA-Seq Kit v4 for Illumina library preparation kit. Please get in touch with us for a custom quote. 

Eukaryotic genes often produce multiple RNA isoforms due to alternative splicing, which can generate functionally distinct protein variants. Nanopore direct RNA full-length transcriptome sequencing directly sequences RNA (with no cDNA conversion!). This has the advantage of long sequencing read lengths which provides better detection of isoforms. This method also directly detects nucleic acid modifications and methylation states of the RNA such as 6 methyl adenine (m6A), 5 methyl cytosine (m5C)and many other methylations/modifications making it quite cutting-edge. Please get in touch with us for a custom quote. 

We use the Parse Biosciences Evercode kits to individually tag each cell in a population (at any scale: from 1000 to 1 million cells or nuclei in a single experiment) so you can get the full transcriptome of each cell in the population. This is useful when you have heterogeneity in your population and want to capture the transciptome of EACH cell. This can also be used on samples after being sorted by flow cytometry (FACS) to further enrich for populations of interest. Please get in touch with us for a custom quote and sample requirements.

Ultra-low input RNA sequencing captures the expression of samples containing as few as 10 pg of RNA or just a few cells. This can be used on samples after being sorted by flow cytometry. We use the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® NEB #E6420S/L for this workflow. This kit uses mRNA enrichment and is not suitable for bacterial samples or samples that lack poly-A. Please get in touch with us for a custom quote.