rRNA Depletion

Total RNA sequencing, or rRNA depletion sequencing, enables the analysis of both coding and noncoding transcripts leading to high confidence observations of features such as allele-specific expression, alternative splicing, and novel transcripts. This approach relies on rRNA depletion to remove off-target transcripts. Illumina’s rRNA depletion kits enzymatically degrade the ribosomal sequences before cDNA synthesis, with oligonucleotide probes used to flag the rRNA content. This negative selection leaves behind all other transcripts for sequencing and is designed for a higher tolerance of lower-quality RNA. It results in increased ribosomal content and a larger proportion of intronic sequencing from nascent transcripts. As this protocol requires homologous probes and high RNA integrity for adequate depletion, it can be less efficient than polyA tail capture depending on probe specificity; however, it is irreplaceable for organisms lacking polyA tailed transcripts and for reliably examining noncoding transcripts.

For rRNA depletion sequencing, SeqCenter uses Illumina’s Stranded Total RNA Prep with Ribo-Zero Plus Microbiome kit to generate unbiased high-quality RNA libraries. The two depletion kits included in this prep are targeted towards major model organisms as well as a broader range of bacteria with clinical significance. Beyond that, SeqCenter is able to design custom depletion probes in partnership with Illumina to ensure optimal depletion in your sample if your project’s organism is not covered within the standard range. For more information, see the depletion section below.

We strongly recommend visiting our overall Illumina RNA services page for additional considerations when planning any RNA experiment and 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)
12M Paired End Reads (1.8 Gbp)
Example uses: Bacteria
25M Paired End Reads (3.75 Gbp)
Example uses: Yeast
50M Paired End Reads (7.5 Gbp)
Example uses: Larger fungi, fruit flies, algae
100M Paired End Reads (15 Gbp)
Example uses: Mouse, human, simple metatranscriptomes
200M Paired End Reads (30 Gbp)
Example uses: Transcriptome assembly, diverse metatranscriptomes
*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.

Depletion, Standard Probe Target Information, and Custom Probes

SeqCenter’s RNA sequencing services with rRNA depletion rely on both Illumina’s proven Ribo-Zero Plus kit and the expanded range Ribo-Zero Plus Microbiome kit to provide depletion for a broad range of common microbial targets and their hosts. Below is more information on the specific targets that these kits are designed against and custom probe options if yours is not closely related any on the lists.

Illumina’s main rRNA depletion is proven efficient against B. cereus, B. subtilis, E. cloacae, E. coli, S. epidermis, as well as the organisms in ATCC MSA-2002 and MSA-2006. Below is a list of the specific design targets for the probes in this kit.

  • Human cytoplasmic rRNAs: 28S, 18S, 5.8S, 5S
  • Human mitochondrial rRNAs: 12S, 16S
  • Human beta globin transcripts: HBA1, HBA2, HBB, HBG1, HBG2
  • Mouse & Rat rRNA: 16S, 28S
  • Gram(-) Bacterial rRNAs: Escherichia coli: 5S, 16S, 23S
  • Gram(+) Bacterial rRNAs: Bacillus subtilis: 5S, 16S, 23S

For more information on the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus, please see Illumina’s data sheet for the prep and depletion kits.

The Microbiome kit expands the capture probe set and was designed against 5S, 16S, and 23S rRNA from diverse microbial species, including common adult and infant gut bacteria, ATCC MSA-2002, MSA-2005, and MSA-2006. Below is a compiled list of the specific targets included in the three ATCC mixes:

  • Acinetobacter baumannii (ATCC 17978)
  • Acinetobacter johnsonii (ATCC 17909)
  • Bacillus pacificus (ATCC 10987)
  • Bacteriodes fragilis (ATCC 25285)
  • Bacteroides vulgatus (ATCC 8482)
  • Bifidobacterium adolescentis (ATCC 15703)
  • Clostridioides difficile (ATCC 9689)
  • Clostridium beijerinckii (ATCC 35702)
  • Corynebacterium striatum (ATCC 6940)
  • Cutibacterium acnes (ATCC 11828)
  • Deinococcus radiodurans (ATCC BAA-816)
  • Enterobacter cloacae (ATCC 13047)
  • Enterococcus faecalis (ATCC 47077)
  • Enterococcus faecalis (ATCC 700802)
  • Escherichia coli (ATCC 700926)
  • Fusobacterium nucleatum (ATCC 25586)
  • Helicobacter pylori (ATCC 700392)
  • Lactobacillus gasseri (ATCC 33323)
  • Lactobacillus plantarum (ATCC BAA-793)
  • Micrococcus luteus (ATCC 4698)
  • Neisseria meningitidis (ATCC BAA-335)
  • Porphyromonas gingivalis (ATCC 33277)
  • Pseudomonas paraeruginosa (ATCC 9027)
  • Rhodobacter sphaeroides (ATCC 17029)
  • Salmonella enterica subsp. enterica (ATCC 9150)
  • Schaalia odontolytica (ATCC 17982)
  • Staphylococcus aureus (ATCC BAA-1556)
  • Staphylococcus epidermidis (ATCC 12228)
  • Streptococcus agalactiae (ATCC BAA-611)
  • Streptococcus mitis (ATCC 49456)
  • Streptococcus mutans (ATCC 700610)
  • Yersinia enterocolitica (ATCC 27729)

For more information on the Microbiome Depletion Kit, please see Illumina’s data sheet for this kit.

Custom depletion probes can be used in place of the Microbiome add-in, and alongside the standard Ribo-Zero Plus set, to ensure optimal depletion for specific organisms. SeqCenter works with Illumina to design custom probes using either an annotated reference genome or specific ribosomal target sequences. Probes are then ordered from IDT and typically arrive within one to two weeks for use.

For additional information on custom probe pools, including a quotation, please contact us.

Because depletion efficiency is greatly affected by RNA integrity, please see our overall Illumina RNA Services Page for additional tips when working with RNA or preparing for an RNA submission.

Go to Top