mRNA Enrichment

mRNA Enrichment rRNA Depletion

PolyA enrichment library preparation positively selects for transcripts with polyadenylated tails and is often referred to as mRNA sequencing. All mRNA and noncoding transcripts with polyA tails are captured via hybridization to polyT oligos on magnetic beads, leaving untailed molecules behind. RNA integrity is of particular importance with this method as degradation of the polyA tail will result in reduced mRNA capture and may affect downstream analysis. As only polyA tailed transcripts will be included in the final dataset, this is the first choice for many eukaryotic studies looking for changes in coding regions.

For this pipeline, SeqCenter uses the Illumina Stranded mRNA Prep kit to generate unbiased RNA libraries. mRNA enrichment is a more cost-effective method and generates greater exonic coverage; however, the major limitation is that it can only be used for organisms with polyA tails. It also requires high-quality RNA and higher minimum sample concentrations.

We strongly recommend visiting our overall Illumina RNA services page for additional considerations when planning any RNA experiment, and we encourage all customers to check out the tips for handling RNA and guidance when assessing sample quality for submission.

Sample Requirements:

  • Samples shipped frozen on dry ice
  • Total volume of 20 µL or more
  • Sample concentration greater than 50 ng/uL
  • DNAse treated (Recommended, not required)
  • RIN > 6 (Recommended measurement, not required)

What You Will Receive:

  • Two fastq files (2 x 150bp) for each sample
  • Sequencing stats summary
  • Materials and methods summary
  • Data shared via Box folder within 2 weeks

Packages & Pricing

Sequencing Package
(Minimum Read Count Per Sample)		 Price*
25M Paired End Reads (3.75 Gbp)
Example uses: Yeast		 $225.00
50M Paired End Reads (7.5 Gbp)
Example uses: Larger fungi, fruit flies, algae		 $270.00
100M Paired End Reads (15 Gbp)
Example uses: Mouse and human		 $350.00
200M Paired End Reads (30 Gbp)
Example uses: Transcriptome assembly		 $500.00
*Orders >48 Samples receive 5% discount

We offer two levels of RNA analysis packages, Basic and Intermediate, which can help our customers to start analyzing their differential expression studies. These packages are a great starting place for burgeoning bioinformaticians, but they will not pick up non-coding RNA and are not intended for metatranscriptomes. For more information, please visit our RNA Analysis services page.

Due to the sensitive nature of RNA and RNA-seq projects, we recommend an approach tailored to a project’s organism and experimental setup that is carried out in a uniform manner soon after harvesting.

For customer convenience, we do offer RNA extraction services using the Zymo Quick-RNA Miniprep Kit, which is broadly applicable but may not be optimal for all sample types or projects. This kit enables us to quickly isolate DNA-free RNA from cell pellets as well as homogenized tissue samples. We recommend sending cell pellets that contain at least 107-108 cells, in which the pellet is sizable and clearly visible in the tube. For tissue samples, we ask that you homogenize them as much as possible to enable successful extractions.

This extraction kit is compatible with ZymoResearch’s DNA/RNA Shield, and it should be noted on orders when samples have been treated with this product. More info on the Quick-RNA Miniprep Kit can be found here.

rRNA Depletion

rRNA depletion enzymatically degrades the ribosomal RNA fragments from a sample by relying on probes targeted at rRNA sequences specific to the organism. rRNA depletion works well for prokaryotes and will capture all RNA molecules over 150 bp that are not degraded.

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Contact:
91 43rd Street, Ste. 250
Pittsburgh, PA 15201
(878) 227-4915

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Full Flowcell:

We offer full flowcell sequencing services for libraries that have been prepared outside of our lab. For each library, we will need to know the loading concentration, phiX spike-in, and run structure. Data is delivered as a complete run folder and can be demultiplexed upon request if a sample sheet is provided.

SARS-CoV-2 Sequencing:

Our SARS-CoV-2 Sequencing services include an assembly on the SARS-CoV-2 reference genome as a scaffold. These packages provide roughly 200Mbp of paired end reads (2×151 bp) as fastq files and the assembly in fasta format.

We require a minimum sample volume of 15 µL of extracted material (or 250µL for VTM samples) and recommend that only samples with Ct values below 35 are sent (because submissions are not measured for viral load before processing). We offer services for both purified RNA and samples in VTM media that require viral RNA extraction. Please see our SARS-CoV-2 Sequencing page for more detailed information.

Microbiome Sequencing:

Please see our Microbiome Sequencing page for package details, including distributables and sample requirements.

Nanopore Sequencing:

Our Oxford Nanopore sequencing packages provide reads equal to the length of input DNA, delivered as a fastq file.

For each sample, we require a minimum volume of 60 µL, and at least 40 ng/µL dsDNA quantified by Qubit or other dye-based method (100 ng/µL by Nanodrop). Variant calling add-on analysis requires an annotated reference file. DNA extraction services are available for only specific categories of microbes.

Please see our DNA Sequencing: Long Read Sequencing page for more detailed information about the package, variant calling analysis, and extraction requirements.

PacBio:

Our PacBio services deliver HiFi reads with an average read length of 8-12kb for standard offerings and up to 25kb for full flowcell runs. All package names are listed by the minimum number of >Q20 reads that will be delivered. Both assembly and annotation are included with all packages.

For each sample, a minimum volume of 60 µL and at least 35 ng/µL dsDNA (quantified by Qubit or other dye-based methods) are required. SeqCenter strongly recommends utilizing our HMW DNA Extraction service for best results; however, we will also accept samples that are shipped on dry ice to avoid DNA integrity issues caused by the shipping process.

Please see our PacBio Sequencing page for more detailed information about our services.

RNA Sequencing:

We offer both rRNA depletion RNA squencing and polyA-tail enrichment mRNA sequencing. Both provide stranded, paired end reads (2x 151bp) as fastq files.

For each sample, we require a minimum volume of 20µL and at least 50ng/µL RNA. We also recommend, but do not require, that samples be DNAse treated and have a RIN score greater than 6. We also offer Basic and Intermediate RNAseq analyses, which require an annotated reference sequence.

Please see our RNA Sequencing page for more detailed information about both our RNA sequencing and analysis packages.

Illumina Sequencing:

The Illumina sequencing packages produce paired end reads (2×151 bp) delivered as fastq files.

For each sample, we require a minimum volume of 30 µL and at least 10 ng/µL dsDNA quantified by Qubit or other dye-based method (20 ng/µL by Nanodrop). Variant calling add-on analysis requires an annotated reference file. DNA extraction services are available for only specific categories of microbes.

Please see our DNA Sequencing: Whole Genome Sequencing page for more detailed information about the package, variant calling analysis, and extraction requirements.

For additional information about variant calling and short read assemblies, please see our Bioinformatics page.

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