Service Description 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 fragments 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 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.
Sample Requirements:
RNA Quality: RIN of 7 (need to be able to identify 18s and 28s rRNA peaks in mammalian samples)
MBCF optimal input : 100-200ng (as low as 50ng)
Volume & amount : >10ul and ~500ng
Analytical Goals:
Gene expression profiling, differential expression testing between conditions (replicates required!), pathway, gene ontology and gene set enrichment analysis
NOT suitable for degraded RNA (e.g FFPE), or analyzing miRNA, or non-polyA non-coding RNAs
Example Method Description: Library Preparation and Sequencing Libraries were prepared using Roche Kapa mRNA HyperPrep strand specific sample preparation kits from [200ng] of purified total RNA according to the manufacturer’s protocol on a Beckman Coulter Biomek i7. The finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final pool was sequenced on an [Illumina NovaSeq 6000] targeting 40 million 150bp read pairs per library at the Dana-Farber Cancer Institute Molecular Biology Core Facilities.