- Sequence Smarter / Ribodepletion RNAseq
Ribodepletion RNAseq
Suitable for prokaryotes or eukaryotes
SeqCoast provides high-throughput Illumina ribodepletion RNA sequencing for microbial isolates, mixed populations, and metatranscriptomes.
Ribodepletion RNAseq
We run NextSeq 2000 and NovaSeq X platforms with X-LEAP chemistry to deliver 2×150 bp paired-end data for differential expression analysis, transcriptomics, and metatranscriptomics, at batch sizes from dozens of samples to tens of thousands of samples.
Why choose ribodepletion RNAseq at SeqCoast?
Probe- and enzyme-based technology removes abundant ribosomal RNA, allowing analysis of other transcripts in your sample.
Our stranded libraries preserve transcript orientation, with 150 bp paired-end reads for detailed RNA analysis.
We run projects from dozens of samples to tens of thousands, across NextSeq 2000 through NovaSeq X.
We use modern X-LEAP chemistry for high read accuracy and high output per run.
Which RNAseq service fits your samples?
Ribodepletion supports prokaryotic, eukaryotic, and mixed samples.
For eukaryotic studies focused on polyadenylated mRNA, explore our Poly-A Enrichment RNAseq service.
Example applications for ribodepletion RNAseq
Example Ribodepletion RNA sequencing targets:
- Prokaryotes (bacteria and archaea)
- Bacterial isolates
- Dual-species samples
- Whole populations (e.g. experimental evolution, intrastrain diversity)
- Metatranscriptomes (e.g. microbiome, soil, contrived communities, wastewater)
- Mixed samples that include prokaryotes and eukaryotes such as human, mouse, and rat - Includes host-pathogen RNAseq/dual RNAseq
Example applications for ribodepletion RNAseq
- Transcriptomics: Accurately identify and characterize gene expression patterns, connecting genomic signatures with active transcriptional patterns.
- Metatranscriptomics: Capture snapshots of gene expression across populations.
- Differential expression analysis
SeqCoast features and benefits
Our expertise and high-throughput capabilities make us the ideal partner for custom projects of any size or complexity, whether you have dozens or thousands of samples.
Flexibility at scale
- High-throughput sequencing, with capacity to process hundreds of thousands of samples.
- Data volumes ranging from 12 million reads to 100+ million reads per sample.
Gold-standard library preparation and sequencing
- Illumina library prep and sequencing reagents. No off-brands, homemade reagents, or compromised quality.
- Broad-range probe and enzyme-based rRNA depletion technology depletes rRNA from a wide range of bacteria, along with humans, mice, and rats.
- Stranded library preparation ensures precise differential expression analysis of overlapping genes and enables detection of antisense transcripts.
- We exclusively use unique dual indexes (UDIs) to prevent index hopping.
- High read accuracy (Q30+) and high throughput.
Multi-point QC to ensure delivery of high-quality data
- All samples checked upon arrival to ensure they have arrived intact and conform to biosafety standards.
- Library concentrations are measured to ensure appropriate library pool balancing and optimize data output for each sample.
- Sequencing run quality metrics checked by a qualified expert.
- Custom QC services available upon request for large orders.
Need custom sequencing?
Most projects are covered by our standard analysis pipelines above. If your project requires custom analysis, tell us about your project goals and needs!
How to prep samples for submission
Raw Samples
- Send raw samples for our RNA extraction workflow, or
- Send between 25 and 200 ng/uL of RNA in a volume of >20ul. We accept samples that have lower concentrations/volumes, but they may have a lower chance of success.
We require:
- We require that you measure your RNA concentrations and dilute to <200 ng/uL.
- We strongly recommend quantifying by Qubit or other fluorometric method, as other methods (such as Nanodrop or Tapestation) are often wildly inaccurate.
Other considerations:
- We recommend eluting samples in nuclease-free water or diluted TE, but we do accept other elution buffers.
- We strongly recommend that you perform a DNAse treatment prior to sending your samples.
- To get the best results, aim for RNA Integrity Numbers (RINs) of at least 7.
- Excessive EDTA and extraction contaminants (DNA, salts, phenol, polysaccharides, etc.) can cause sample failure.
48+ samples
- We offer our COMPASS system for CoOrdinated Management and Processing for Accelerated Sequencing Services.
- COMPASS allows for the fastest processing and the lowest price!
- You will receive a custom shipping container with validated plasticware, stabilization buffer, & prepaid shipping label at no additional charge.
24-48 samples
- Must be sent in a 96-well plate or individually-capped 8-tube strips.
- 96-well plates should be fully-skirted and sealed well with a foil seal (plastic seals are very prone to leaking during shipping).
<24 samples
- Individually-capped 8-tube strips (preferred) or 1.5 mL tubes.
- Other sample formats may cause delays and additional fees.
96-well plates
- Label with Order Number and (if necessary) Plate Number.
Tubes
- Label caps with SeqCoast Sample ID (assigned in your Sample Manifest).
- Labeling tubes with your own sample names instead of the SeqCoast Sample IDs can cause delays!
Please send
All samples must be securely packaged in a clean, organized fashion using secondary containment. Leaking samples will be immediately disposed of, as required by our biosafety measures.
Please do not send
Bags of tubes, individual tubes wrapped in parafilm, large-format tubes (e.g. 50ml conicals), non-sterile/soft-sided containers (e.g. plastic bags), unsealed cell culture 96- well plates. We’ve seen it all! Please double-check your shipments to ensure your samples make it to us safe and sound.
Data delivery, security, and access
We make it easy to access your data!
Data delivery options include SFTP Download, AWS transfer, Box.com, and alternatives on request.
Most RNAseq orders are completed within 14 days of sample receipt.
Data are stored on AWS cloud infrastructure in the United States, providing scalable storage, high availability, and industry-recognized security controls.
Pricing — the more you order, the lower the price!
How much sequencing data do I need for RNAseq?
If you are unsure about the data volume per sample you need, we recommend that you do one of the following:
- Perform a literature search for similar sample types and analyses.
- Send pilot samples aiming for more data than you think you need, then use down-sampling methods to determine the optimal data volume for your full experiment.
While we do not provide specific recommendations for every project, please see the following table for illustrative examples of data volume tiers that our customers have used:
Frequently Asked Questions
How should I extract my RNA?
Let us do the hard work for you! We offer RNA extraction using bead beating.
If you want to tackle it yourself, many column-based extraction kits provide good results, as do other methods if care is taken to remove contaminants.
Be conscious of the timing of extraction. Transcription activation can be fast, and for some experimental conditions, even minutes matter. Save some heartache down the road by thoughtfully considering the logistics at the start.
DNase treat your samples. For best results, DNA should be low or undetectable. In some cases, multiple DNase treatments may be required.
Can I send a lower concentration or volume than your recommendations?
Yes, we are often successful with lower-concentration samples and are happy to process any samples that you send!
Please note that samples that do not meet our recommendations have a lower chance of success.
Custom low-input library preparation services are available for large orders.
What is RIN and do you have RIN requirements?
RNA Integrity Number (RIN) is a measure of RNA degradation on a scale of 1 (worst) to 10 (best).
RIN can be estimated using agarose gels, but is more precise on a Bioanalyzer, Tapestation, or similar instrument.
We generally recommend that RINs be at least 7, but we can process any sample you send us.
Depending on your project goals, lower RINs may still provide adequate results.
Please note that we do not measure RIN as part of our standard QC process, so we recommend that you check it prior to sending your samples.
Is RNAseq a coverage or counting application?
RNAseq is a counting application. In other words, it surveys the relative abundance of specific sequences. Therefore, data quantity is measured in terms of number of reads sequenced, not by genome coverage.
This contrasts with coverage applications, for which data quantity is measured in terms of the average depth of sequencing across the entire genome.
How much sequencing data do I need?
See the “How Much Sequencing Data Do I Need for RNAseq?” section above.
How many biological replicates do I need?
At least three biological replicates are required for differential expression analysis.
For projects looking at subtle transcriptomic changes or with “noisy” samples, we recommend more than three biological replicates.
How many transcripts will be “counted” by a given number of reads?
Paired reads “count” the same sequence.
For example:
- 12M total reads count 6M transcript fragments
- 50M total reads count 25M transcript fragments
Will the ribodepletion work for my species?
We use Illumina’s gold-standard Ribo-Zero Plus Microbiome rRNA depletion kit, which depletes abundant transcripts from the following targets:
- Wide range of bacteria and other microbes
- Human cytoplasmic and mitochondrial rRNA
- Human beta globin transcripts
- Mouse and rat rRNA
If you are unsure whether your species will be compatible, then we recommend placing a small pilot order to check whether the ribodepletion is successful.
Have a large sample quantity or long-term, multi-batch project? Get in touch with our team to see if your project qualifies for a complimentary project pilot!
Is the library preparation stranded?
Yes! All of our RNAseq library preparation methods preserve strand information by distinguishing between the first and second cDNA strands.
Stranded library preparation ensures precise differential expression analysis of overlapping genes and enables detection of antisense transcripts.
Not all providers use this method!
Do you have requirements for A260/230 and A260/280 absorbance ratios?
Our library preparation workflows are generally quite robust and can tolerate moderate deviations from ideal absorbance ratios.
We do not have requirements surrounding absorbance ratios and will not provide recommendations on whether your ratios are sufficient.
However, please be aware that lower-quality samples have lower chances of success and may require that you perform a cleanup prior to sending them for sequencing.