TotalRNAseq (rRNA Depletion)
Service Description
Ribosomal RNA (rRNA) represents >80% of cellular RNA. This “TotalRNAseq” method takes purified DNase treated total RNA as input and removes the rRNA fraction. The MBCF has experience with a number of different kits and protocols, but we have adopted Qiagen FastSelect rRNA depletion reagents and protocol as our preferred method. This proprietary method uses rRNA specifc probes to block reverse transcription of rRNA sequences during library prep resulting in a fast and efficient means for rRNA removal. Subsequent Illumina library prep is performed on a Biomek i7 automated liquid handler using Roche Kapa RNA Hyper Prep reagents.
Sample Requirements:
- RNA Quality: DV200 > 40%
- DNase Treated purified RNA
- 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
- suitable for degraded RNA (e.g FFPE) and non-polyA non-coding RNAs
Other Information:
- Strand specific
- Receive 50M 150bp read pairs per sample
- Works with DNA contamination
- starting volume is 50uL
Recommended Bioinformatics Pipeline: Viper (Visualization Pipeline for RNAseq)
Example Method Description
Library Preparation and Sequencing
rRNA depletion was performed from [100ng] of purified RNA using QIAseq FastSelect rRNA HMR reagents according to manufacturer’s protocol. Libraries were prepared using Roche Kapa Biosystems RNA HyperPrep sample preparation reagents on a Beckman Coulter Biomek i7. 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 with paired-end 150bp reads on an Illumina NovaSeq X Plus at the Dana-Farber Cancer Institute Molecular Biology Core Facilities.
Acknowledgements (if sequenced with NovaSeq X Plus):
This work utilized an Illumina NovaSeq X Plus that was purchased with funding from a National Institutes of Health SIG grant 1S10OD036228-01.