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Genomics Core

Library Preparation

Library Preparation

RNA + DNA Library Preparation

The Genomics Core offers full library prep services for many sample types and applications for the Illumina HiSeq and Miseq platforms. If the library prep that is of interest to you is not listed below, please contact the Core Manager, Simone Edelheit to discuss your project.

Sample Submission Requirements

  • Submit all samples in 1.5ml tubes, clearly labeled ON TOP OF TUBE with sample name and user name/initials (sample name on request form and on tube should match). Label the SIDE OF THE TUBE with the date.
  • Large volume projects may be submitted in FULL SKIRT 96-well plates, with at least one SIDE WALL clearly labeled with project name, plate number, user name, and date. Sample names and well location will be submitted through iLab. 
  • For specifics on total amount and volume required, please see RNA and DNA library preparation options in the charts below. 
  • To begin a request for service please follow the iLab instructions:

The Genomics Core is now transitioning all service requests over to iLab Operations Software, a part of Agilent Technologies’ CrossLab platform. This is an online system to streamline the process of ordering and billing for core service requests. 

In order to gain access to your funds you will need to begin by logging into iLab at the following web address:

https://cwru.corefacilities.org

Once an account is created, the Genomics Core ordering site can be found here:

https://cwru.corefacilities.org/service_center/show_external/4142?name=genomics-core-facility

Simply click on Request Services tab to begin. 

 

RNA Library Preparation Options

All RNA submitted to the Core for library processing will undergo quality control (QC) to make sure it is of high enough quality and quantity to move forward with library preparation. The QC of each sample includes Qubit for concentration (ng/ul) and the Agilent Bioanalyzer RNA chip to determine the RNA Integrity Number (RIN). The cost of the QC is included in the price of library prep. If a sample fails QC, the user will be charged for QC portion only. The Core will provide expert advice as to whether or not an RNA sample should be used for downstream analysis, but the final decision will be made by the user. Please see User Agreements page and the NGS Run Guarantees Section.

Since 2015, the Core has partnered with Dr. Mark Cameron's lab, from the Department of Population and Quantitative Health Sciences, to provide RNAseq analysis from ultra-low RNA input amounts. Contact the Core for more details. 

  • For a list of available preps and pricing, please see Pricing.
     
RNA Library Prep KitDescriptionAmount Strongly Recomended Amount Required (per Qubit)Typical Run Type & Coverage
Illumina TruSeq Stranded Total RNA w/ Ribo Zero Gold
  • Whole-transcriptome analysis
  • Generates directional RNA-focused libraries
  • Low-quality and FFPE compatible
  • Removes both cytoplasmic and mitochondrial rRNA
  • Up to 96 unique indexes
  • Use with standard input RNAs (>15ng/ul)
  • 50ng/ul in ≥20ul
  • RIN: 7.0 - 10.0 

     

  • 15ng/ul in 20ul
  • RIN: 2.0-10.0
  • HiSeq 1x50bp for Gene Expression only
  • HiSeq 2x100bp for Alternative Splicing
  • 50M Paired-reads per sample
Low and Ultra-low RNA library Prep
  • The Core and user will work directly with Dr. Mark Cameron's Lab
  • TBD
  • TBD
  • TBD
Illumina TruSeq Targeted RNA Expression Kit (TREx) (custom kits and fixed panels)
  • Study up to 15,000 gene expression results using 50bp sequencing reads to target specific exons, splice junctions, cSNPs, and gene fusions
  • Fixed panels also available include Apoptosis, Cardiotoxicity, Cell Cycle, Cytochrome P450, Hedgehog Pathway, Neurodegeneration, NFB Pathway, Stem Cell, TP53 Pathway, & Wnt Pathway
  • Supports 384 samples per run and 12–1000 assays per sample
  • Automated final data analysis using the Illumina MiSeq software
  • 20ng/ul in ≥20ul 
  • RIN: 7.0 - 10.0
  • 5ng/ul in 20ul
  • RIN: 2.0-10.0
  • MiSeq v3 1x50bp
  • Work with Illumina Sales Representative for ordering custom project.

DNA Library Preparation Options

All DNA submitted to the Core for processing will undergo quality control (QC) to make sure it is of high enough quality and quantity to move forward with library preparation. The QC of each sample includes Qubit for concentration (ng/ul) and agarose gel electrophoresis to check quality. The cost of the QC is included in the price of library prep. If a sample fails QC, the user will be charged for QC portion only. The Core will provide expert advice as to whether or not a DNA sample should be used for downstream analysis, but the final decision will be made by the user. See User Agreements page and NGS Run Guarantees section for more information.

  • For a list of available preps and pricing, please see Pricing.
 
DNA Library Prep KitDescriptionRequirements   (per Qubit)Typical Run Type & Coverage
Illumina TruSeq DNA PCR Free
  • WGS of small genomes to whole-human genome sequencing Shortened GEL-FREE workflow for reduced library bias and gaps
  • Up to 96 unique indexes
  • 100ng/ul in 20ul
  • 260/280: 1.8-2.0
  • HiSeq 2x100bp
  • MiSeq v3 2x250bp
Nextera XT DNA
  • Prepare libraries for small genomes, PCR amplicons, and plasmids to sequence using MiSeq technology Multiplex up to 384 samples
  • 10ng/ul in 20ul
  • 260/280: 1.8-2.0
  • MiSeq v3 2x250bp
Nextera Rapid Capture Exome
  • All-in-one kit for library prep and exome enrichment
  • Includes 37Mb of expertly selected exonic- content
  • Up to 24 unique indexes
  • 10ng/ul in 20ul
  • 260/280: 1.8-2.0
  • HiSeq 2x100bp
  • 100X coverage = 80M Paired reads per sample
TruSeq Exome / TruSeq Rapid Exome
  • All-in-one kit for library prep and exome enrichment
  • Includes 45Mb of exonic content
  • Up to 12 unique indexes 
  • TruSeq Exome is FFPE compatible. 
  • 10ng/ul in 20ul
  • 260/280: 1.8-2.0
  • HiSeq 2x75bp
2-Step PCR Amplicon Approach (CRISPR)
  • Best used for CRISPR/Cas gene editing validation
  • Tailed amplicon sequencing using Nextera Dual indexing adapters and the MiSeq platform
  • Up to 192 unique indexes available. 
  • 10ul PCR product
  • One PCR per well
  • Gel Image
  • MiSeq v3 2x250bp
16S Metagenomics (rRNA Sequencing)
  • Amplicon sequencing method used to identify and compare bacteria present within a given sample
  • Primer pair sequences for the V3 and V4 region that create a single amplicon of approximately ~460 bp
  • Up to 384 unique indexes. 

Either:

  • DNA – 10ng/ul in 20ul
  • V3/V4 PCR product – 25ul w/ gel image
  • MiSeq v3 2x250bp
  • ~100,000 reads per sample
Illumina TruSeq Custom Amplicon Kit
  • Fully customizable, amplicon-based assay for targeted resequencing
  • Multiplex up to 1536 amplicons per reaction to cover up to 650 kb of cumulative sequence
  • Pool up to 96 samples for maximum benefit from the MiSeq v3 reagents
  • Access automated, design-specific variant calling and final data analysis using the Illumina MiSeq software
  • FFPE DNA compatible
  • 10ng/ul in 20ul
  • 260/280: 1.8-2.0
  • Illumina QC Kit required for FFPE samples
  • MiSeq v3 2x250bp
  • Custom analysis workflow provided for MiSeq run.
  • Work with Illumina Sales Representative for ordering custom project.