Targeted RNAseq (cDNA Hybrid Capture)
Service Description
Sample Requirements:
- RNA Quality: DV200 > 40%
- MBCF optimal input : 100-200ng (as low as 50ng)
- Volume & amount : >10ul and >100ng (more is always better)
Analytical Goals:
- Gene expression profiling, differential expression testing between conditions (replicates required!), pathway, gene ontology and gene set enrichment analysis
- Suitable for degraded RNA (e.g FFPE)
Other Information:
- Strand specific
- Twist RNA exome capture
- Receive 40M 150bp read pairs per sample
Recommended Bioinformatics Pipeline: Viper (Visualization Pipeline for RNAseq)
Example Method Description
Library Preparation and Sequencing
Purified total RNA was evaluated for quality and quantity using the Agilent Fragment Analyzer RNA assay. RNA samples were prepared using Roche Kapa RNA Hyper reagents without any prior enrichment or depletion according to the manufacturer’s protocol on a Beckman Coulter Biomek i7. Following library preparation, samples were characterized by Qubit fluorometer and Agilent TapeStation 4400. Libraries were pooled in an [8plex] reactions and hybridized to Twist Exome 2.0 probes for target enrichment. Post capture library pools were quantified by Qubit fluorometer and Agilent TapeStation 4400. Uniquely dual indexed libraries will be pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final library pools were sequenced on an Illumina NovaSeq X Plus targeting with 150bp paired-end reads at the Dana-Farber Cancer Institute Molecular Biology Core Facilities.
Acknowledgements (if sequenced with NovaSeq X Plus):
This work utilized an Illuimina NovaSeq X Plus that was purchased with funding from a National Institutes of Health SIG grant 1S10OD036228-01.