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.