RNA-Seq

Plasmidsaurus RNA-Seq provides 3’ end counting for transcriptome-wide differential gene expression data. We utilize the Illumina platform to provide ~10M deduplicated 3' end counting reads (20M raw reads) from ~100k cells or 300 ng purified RNA, returned in ~3 days.

Every order includes interactive data analysis tools on the results page. These tools enable immediate insights into your experiments, providing actionable data analysis within minutes of getting your results.

There is no minimum order size or quote process. Cell samples do not require cold chain shipping.

The gold standard method to determine the accuracy of any RNA-Seq method is to spike in 92 synthetic RNA sequences at known abundance, designed by the consortium for RNA sequencing (ERCC). We spiked those sequences into 100 ng of universal human reference RNA at a standardized concentration, and then performed Plasmidsaurus RNAseq. Plotting the true concentration against measured abundance reveals that we can accurately quantify true transcript abundance across multiple orders of magnitude of RNA abundance, down to at least 1 CPM.

The goal of Plasmidsaurus RNA-Seq is to accurately quantify transcript abundance. Because PCR creates copies, we use unique molecular identifiers (UMIs), which are added during cDNA creation. This allows us to differentiate between a multitude of unique mRNA molecules versus a molecule that was amplified.

Clustering by UMI sequence allows us to analyze and return deduplicated reads only: this is the most accurate way of quantifying transcript abundance.

We utilize 3’ end counting for our RNA-Seq service, where we utilize the 3’ end of the transcript for gene identification. As such, the current service is unable to distinguish transcript isoforms. 

At this time, we are accepting the following sample types:

• Preserved animal cells: This includes adherent, suspension, and primary cell cultures. For best results, cells should be healthy, actively growing, and free from contamination.
• Purified RNA: We can accept purified bulk RNA, free from DNA and RNAse activity, shipped with dry ice (Continental U.S. only at this time.) We need at least 30μl of at least 10ng/μl concentration.

We are currently NOT accepting the following sample types:

• Non-animal cells: We can’t currently accept plant, fungal, or prokaryotic cells.
• Tissue Samples: We are unable to process tissue samples at this time, but we look forward to offering this in the near future.

Please send ~100k cells in 50 μL of 1X Zymo DNA/RNA Shield, either through our extensive network of drop boxes, or via direct shipping at room temperature—no cold chain required. If you’re submitting purified RNA, we cannot provide free shipping at this time. For all the details on sample input guidance, please see here.
 

Biological replicates are not required, but strongly encouraged in order to get the most statistical power for your analysis.

Library preparation contains the following steps:

1. Poly-A mRNA capture and reverse transcription for cDNA synthesis: oligo-dT primer to capture just the mRNA (contains sample barcode, UMI, and Read 1 sequences).
2. Second strand synthesis and tagmentation: double stranded-cDNA is generated. Tagmentation creates fragments and incorporates Read 2 sequence.
3. Illumina library amplification: adds unique dual indices (UDI = i5, i7) and P5/P7 sequences.

You will receive an email once your samples have been sequencing and the data is ready. For RNA-Seq we provide an online report with data analysis tools as well as a downloadable package.

Our online report provides you the ability to interact with volcano plots, functional enrichment pathway diagrams, and charts of gene expression by sample and/or sample groups. 

If you would like to dig in further, we provide the following content in a downloadable package: quality control summary (you will also receive QC info like the fastqc) summary, aligned deduplicated bam, and gene counts. You will also receive the raw fastqs.

Our interactive results page lets you explore the most differentially represented genes across your samples, create and explore differential gene expression analyses (think Volcano Plots), detect enriched cellular pathways, select genes to track across conditions, and more. Pathway and gene count comparisons are all exportable.

If you’d like to do your own offline analysis, we provide the following downloadable content: quality control summary (fastqc), aligned deduplicated bam files, and gene counts in structured plain text format. You can also download the raw fastqs.

Occasionally samples will produce substantially fewer reads than expected. This can happen for many reasons, such as a shortfall in the number of cells submitted, lower than estimated transcriptional activity, very low RNA integrity, or some issue during processing after receipt of your sample. 

If we detect a failure in our process, we will attempt to rerun your sample, provided there is sufficient input material. You can help improve the probability of success for that by submitting higher sample amounts (towards the upper end of our input guidance) when your experimental design allows that without hardship on your end.