mRNAseq
This RNA library preparation method takes total purified RNA as input and enriches for polyadenylated (polyA+) transcripts using oligo-dT coated beads. PolyA+ RNA is subsequently fragmented using heat and Mg+ and reverse transcribed into cDNA using random priming. Illiumina adapters are ligated to dsDNA and PCR amplified.
The MBCF has worked with several different mRNAseq library prep kits including Illiumina, Roche Kapa, and NEB. Our default method which we determined to be the most sensitive, robust, cost-effective, and high throughput (automated on Biomek FX and i7) method in our hands is Roche Kapa mRNA Hyper Prep. We are willing and able to use another kit if needed – may increase turnaround time.
Pricing
QC + library prep + sequencing (40M 100bp read pairs)
DFCI/BWH: $340
External: $400
RNAseq analysis (VIPER)
DFCI/BWH: +$20/sample
External: +$25/sample
Turnaround
3-4 weeks
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RNA QC
All Samples are assayed by Agilent Bioanalyzer or Fragment Analyzer to evaluate RNA quality and quantity. If any samples do not meet our requirements, you will be contacted by core staff with experience based recommendations along with an opportunity to replace any problematic samples is possible.
Analytical Goals
Gene expression profiling, differential expression testing between conditions (replicates required!), pathway, gene ontology and gene set enrichment analysis.
NOT a suitable method for degraded RNA (e.g. FFPE) or analyzing miRNA, or non-polyA non-coding RNAs
Sample Requirements
RNA Amount: 500ng (more is fine, less can be OK too)
RNA Quality:RIN/RQN > 7
Common Extraction Methods: Qiagen (i.e. RNeasy, miRNeasy) or Trizol. DNase treatment strongly recommended.
Minimum Input: 50ng
MBCF Optimal Input Amount: 100-200ng (for Kappa)
Volume: >10ul
Concentration Range: 5-500 ng/ul (it will depend)