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Strand-specific bulk mRNA sequencing quantifies gene expression and resolves differentially expressed genes. It supports research into cell growth, tumorigenesis, and immune response, with applications spanning cancer research (including prevention and treatment strategies), disease-specific gene expression studies, and RNA drug response biomarker investigations. Library preparation uses commercially available kits from Illumina, Watchmaker Genomics, Takara Bio and others.
For read depth recommendations, refer to the FAQ section on this page.
For large scale gene expression screens, SeqMatic offers 3′ RNA sequencing, a high throughput approach that sequences the 3′ end of each transcript rather than its full length. Because only the region near the poly-A tail is read, far fewer reads are needed to quantify gene expression accurately, which makes 3′ RNA sequencing well suited to studies that compare expression levels across many samples, such as compound and CRISPR screens, dose-response and time-course experiments, and large cohort studies.
Standard bulk mRNA-seq and 3′ RNA sequencing answer different questions:
Because reads concentrate at transcript ends, 3′ RNA sequencing is not suited to alternative splicing, isoform quantification, fusion detection, variant calling, allele-specific expression, or novel transcript discovery. For those conditions standard mRNA-seq remains the better choice for those endpoints.
Total RNA sequencing with ribosomal depletion (RiboZero+) enriches for informative transcripts rather than relying on poly-A selection. It is the preferred method for transcriptomic analysis of whole blood, cells such as bacteria, and tissues such as spleen, where poly-A mRNA is either unavailable or overwhelmed by globin RNAs, and it is often used for poor quality samples such as FFPE. Total RNA-seq detects coding transcripts plus multiple forms of non-coding RNA, including structural, long non-coding, small, and miRNAs, and provides optimal coverage of normal or low quality samples. The standard library preparation is Illumina Total RNA with RZ+; alternate kits available on request.
Small RNA (miRNA) sequencing profiles short, non-coding RNA from any organism without the need for reference sequences. Applications include biomarker discovery, miRNA expression profiling, and detection of rare transcripts. SeqMatic’s founders are former Illumina scientists who developed the Illumina small RNA kits, and the SeqMatic TailorMix miRNA kit delivers strong results from difficult samples such as blood, plasma, and saliva. The service accepts as little as 1 ng of total RNA or purified miRNA. Small RNAs are isolated by size using gel electrophoresis and gel purification, a hands-on size-selection step that relatively few service providers offer, followed by 50 bp single-end sequencing on Illumina platforms.
FFPE RNA sequencing enables gene expression profiling of formalin fixed paraffin embedded tissue, and is used in studies of disease progression and in identifying biomarkers for diagnosis and treatment. This service covers bulk total RNA sequencing from FFPE material; working with degraded FFPE RNA is a specialty that not all providers offer. Single cell and spatial profiling from FFPE are also available and are covered on the single cell RNA sequencing page.
SeqMatic’s recommended FFPE library preparation is the Watchmaker Genomics RNA Library Prep Kit with Polaris Depletion, which is built for degraded and low input material. It depletes ribosomal and globin transcripts to improve coverage of biologically informative transcripts, including long non-coding RNA, and accepts inputs from 1 ng to 1,000 ng of total RNA. A dedicated FFPE treatment step, paired with a reverse transcriptase engineered for RNA-seq, recovers usable signal from cross-linked and damaged FFPE RNA where standard workflows lose sensitivity.
If FFPE blocks are provided, SeqMatic offers a full service histopathology lab to section the blocks to support the RNA-seq workflow.
RNA quality is assessed by TapeStation before library preparation. Recommended read depth and input requirements for FFPE are given in the tables below.
Exosome RNA sequencing characterizes the comprehensive RNA content of exosomes, covering both protein coding and long non-coding RNA transcripts, to demonstrate the RNA cargoes carried within exosome populations released by different cell types. Exosomes and other extracellular vesicles are found in biofluids including saliva, blood, urine, and cerebrospinal fluid, and their non-coding RNAs are believed to be key regulators of molecular activity in recipient cells. This specialty service uses gel cut purification and size selection, with a typical read depth of 30 million reads per sample and higher or lower depths available depending on project needs.
Every project begins with a scientific consultation. The team learns the goals of your research, then modifies the RNA workflow and kit selection to target those goals, recommending the appropriate library preparation and sequencing depth. For challenging samples, the team works with you on the best approach before submission, drawing on documented experience with low input material (small RNA workflows accept as little as 1 ng), degraded and FFPE tissue, and non-model organisms.
For researchers in the San Francisco Bay Area, Fetch-Seq is a dedicated, door to door courier pickup service that retains full chain of custody and real-time tracking for transport of all sample types. Samples are delivered directly to SeqMatic, typically within 1 to 1.5 hours of pickup depending on location, as a personalized pickup rather than a shared route with fixed stops, covering a 100 mile radius from Fremont, CA. The benefit is simple: time sensitive and fragile RNA samples reach the lab quickly and with a documented handoff, reducing the degradation and chain-of-custody gaps that shipping can introduce.
SeqMatic accepts samples at any stage of the workflow, and accepts a wide range of sample types: extracted nucleic acids, tissue (animal and plant), cells, whole blood, plasma, serum, soil, fecal and environmental specimens, FFPE blocks and sections, and cryopreserved material. Detailed input requirements by library type are given in the sample requirement tables below.
If a sample requires nucleic acid extraction, this is performed in-house as the first step of the workflow, using established vendor kits selected by sample type, including QIAGEN, Zymo Research, Promega, Macherey-Nagel, and Roche. If a customer submits purified RNA, SeqMatic recommends it be stored in nuclease free water or an RNA preservative reagent and, if required, validated on a TapeStation electropherogram, which reports RNA Integrity Number (RIN) and DV200.
SeqMatic qualifies each RNA sample before library preparation and shares the results for review and approval. If a sample does not meet the requirements, the team consults with the customer on how best to proceed.
Library preparation is matched to the RNA species being profiled, and each workflow is distinct:
All sample preparation and library construction steps are validated by multiple assays (TapeStation, qPCR, and NGS test sequencing) under proven, validated SOPs. After preparation, libraries are pooled to customer requirements, and the final pool is QC checked via preliminary sequencing on MiSeq, then normalized and re-pooled based on the read counts from that preliminary run to ensure equal coverage across samples. SeqMatic’s proprietary TailorMix dual indexed PhiX control library supports data processing by reducing cross-talk between samples.
Short-read RNA libraries are sequenced on Illumina platforms, including MiSeq and NextSeq for QC and smaller projects and NovaSeq 6000 and NovaSeq X Plus for production scale sequencing. For full length transcript and isoform-level analysis, long-read RNA sequencing is available on the PacBio platform with the Kinnex Library Prep Kit. Platform selection for each project is matched to the number of samples, read depth, and read configuration.
SeqMatic provides a full suite of custom bioinformatics pipelines that can be tailored to each project, including support for non-model and poorly annotated genomes. Analysis is offered at three tiers, and the pipeline can go deeper per customer request:
Standard RNA-seq projects are completed in 4 to 6 weeks from sample receipt to data delivery, covering extraction, sample QC, library preparation, sequencing, and primary data analysis. Express turnaround is available in as few as 2 to 3 weeks for time sensitive projects. RNA-seq screening studies can be completed in 5-10 days. Actual turnaround depends on sample quality, the number of samples, sequencing depth, and the scope of bioinformatics analysis requested.
| Application | Library Type | Recommended Depth |
|---|---|---|
| Standard transcriptome analysis | Bulk mRNA-Seq | 25 to 30 million reads per sample |
| RNA screening | 3′ RNA-Seq | 2 to 10 million reads per sample |
| Total transcriptome analysis | Total RNA-Seq | 50 to 60 million reads per sample |
| FFPE expression profiling | FFPE Bulk Total RNA-Seq | 50 to 100 million reads per sample |
| Exosome RNA profiling | Exosome RNA-Seq | 30 million reads per sample |
| Small RNA / miRNA | miRNA & Small RNA | 10 million reads per sample |
| Parameter | Recommended | Minimum |
|---|---|---|
| Purified RNA amount | 1 µg (1000 ng) or more | 25 ng (discuss with a SeqMatic scientist if lower) |
| Volume | 20 µL or more | 10 µL |
| Concentration | 20 ng/µL or higher | 5 ng/µL (discuss if lower) |
| Purity (A260:A280) | 1.8 to 2.0 | 1.8 to 2.0 |
| Quality | RIN >8 | RIN >7 |
| Parameter | Recommended | Minimum |
|---|---|---|
| RNA amount | 1 µg (1000 ng) or more | 1 ng (discuss with a SeqMatic scientist if lower) |
| Volume | 20 µL or more | 10 µL |
| Concentration | 20 ng/µL or higher | 1 ng/µL (discuss if lower) |
| Purity (A260:A280) | 1.8 to 2.0 | 1.8 to 2.0 |
| Quality | RIN > 7 / DV200 > 55% | DV200 > 35% |
| Parameter | Recommended | Minimum |
|---|---|---|
| RNA amount | 1 µg (1000 ng) or more | 10 ng (discuss with a SeqMatic scientist if lower) |
| Volume | 20 µL or more | 10 µL |
| Concentration | 20 ng/µL or higher | 1 ng/µL (discuss if lower) |
| Purity (A260:A280) | 1.8 to 2.0 | 1.8 to 2.0 |
| Quality | RIN > 7 / DV200 > 55% | DV200 > 35% |
| Parameter | Recommended | Minimum |
|---|---|---|
| Purified RNA amount | 500 ng or more | 1 ng (discuss with a SeqMatic scientist if lower) |
| Volume | 20 µL or more | 10 µL |
| Concentration | 20 ng/µL or higher | 1 ng/µL (discuss if lower) |
| Purity (A260:A280) | 1.8 to 2.0 | 1.8 to 2.0 |
| Special requirements | Protocol must retain small RNAs >15 nt | Protocol must retain small RNAs >15 nt |
RNA sequencing measures gene expression averaged across all the cells in a sample, producing one expression profile per sample. Single cell RNA sequencing measures expression in individual cells, resolving cell type composition and rare populations that an average cannot show. RNA-seq is the right choice for population level comparisons, limited or degraded input material, and high throughput studies; single cell RNA-seq is the right choice when cellular heterogeneity drives the question. Many studies use both.
Choose total RNA-seq with ribosomal depletion when poly-A selected mRNA-seq would miss the transcripts you need. It is preferred for whole blood, cells such as bacteria, and tissues such as spleen, where poly-A mRNA is unavailable or overwhelmed by globin RNAs, and it is often the better option for poor quality or degraded samples such as FFPE. Total RNA-seq retains coding plus non-coding, structural, small, and miRNA transcripts. Choose mRNA-seq when your focus is protein coding gene expression and your RNA is intact.
Input requirements depend on the library type; see the four sample requirement tables above for recommended and minimum values. As a general guide, standard mRNA-seq recommends 1 µg of total RNA, and SeqMatic’s low input and small RNA workflows accept far less. If you cannot meet the minimum for your library type, discuss it with a SeqMatic scientist before submission, since lower input is often feasible.
SeqMatic qualifies each submitted RNA sample on a TapeStation electropherogram, which reports RIN and DV200. Recommended and minimum RIN and DV200 values by library type are given in the sample requirement tables above. Results are shared with the customer for review and approval before library preparation proceeds, and if a sample falls below requirements the team consults on how best to proceed rather than failing it automatically.
Yes. SeqMatic provides bulk total RNA sequencing from FFPE material. The recommended FFPE library preparation is the Watchmaker Genomics RNA Library Prep with Polaris Depletion, which combines ribosomal and globin depletion with a dedicated FFPE treatment step to recover signal from degraded, cross-linked RNA, with inputs from 1 ng to 1,000 ng. Recommended depth for FFPE bulk total RNA-seq is 50 to 100 million reads per sample. RNA quality is assessed by TapeStation before library preparation, and a full service histopathology laboratory is available to section FFPE blocks. Single cell and spatial FFPE profiling are covered on the single cell RNA sequencing page.
SeqMatic recommends 25 to 30 million reads per sample for standard mRNA-seq and 50 to 60 million for total RNA-seq, 50 to 100 million for FFPE bulk total RNA-seq, 30 million for exosome RNA-seq, 10 million for small RNA / miRNA, and 2 to 10 million reads for 3′ RNA screening. These figures apply to model organisms such as human and mouse; other species may differ, and higher or lower depths are available depending on project needs.
As a guide: choose Bulk mRNA-Seq for protein coding gene expression from intact RNA; choose Bulk Total RNA / Non-coding RNA for whole blood, bacterial, degraded, or FFPE samples, or when you need non-coding RNA coverage; choose miRNA & Small RNA for microRNA and other small non-coding RNA; choose FFPE RNA for archival tissue; choose Exosome for RNA cargo from biofluids; and consider 3′ RNA sequencing for large screens across many samples. SeqMatic’s team helps confirm the right package during the project consultation.
Yes. SeqMatic provides custom bioinformatics at three tiers — Primary (alignment and quantification), Secondary (QC, normalization, and differential expression), and Tertiary (pathway, gene ontology, and functional interpretation) — and can go deeper per customer request, including support for non-model organisms.
The SeqMatic approach starts with proven genomic workflows and proprietary controls that have been refined and optimized over the past decade to deliver considerably more data than Illumina specifications.
The SeqMatic approach starts with proven genomic workflows and proprietary controls that have been refined and optimized over the past decade to deliver considerably more data than Illumina specifications.
View More We learn about the goals of our customer’s scientific research and the type of sequencing data that would be most beneficial to their project.
We learn about the goals of our customer’s scientific research and the type of sequencing data that would be most beneficial to their project.
View More Then we modify our workflow to target those specific reads. Couple that effort with world class turnaround time.
Then we modify our workflow to target those specific reads. Couple that effort with world class turnaround time.
View More This application of best science, higher quality data, more read depth per lane, and fast delivery makes SeqMatic an exceptional partner and value per research dollar spent.
This application of best science, higher quality data, more read depth per lane, and fast delivery makes SeqMatic an exceptional partner and value per research dollar spent.
View MoreOptimal solutions are custom-made according to research needs
