Low Input mRNAseq (polyA Enrichment)
Service Description
Sample Requirements:
- RNA Quality: RIN of 7 (need to be able to identify 18s and 28s rRNA peaks in mammalian samples)
- MBCF optimal input : 500pg - 10ng
- Volume & amount : >10ul
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
Other Information:
- Non-stranded
- Receive 40M 150bp read pairs per sample
- Can tolerate minimal DNA contamination (DNase treatment still recommended)
Recommended Bioinformatics Pipeline: Viper (Visualization Pipeline for RNAseq)
Example Method Description
Library Preparation and Sequencing
cDNA was synthesized using Takara SmartSeq v4 reagents from [1ng] of RNA. Full length cDNA was fragmented to a mean size of 200bp with a Covaris R230 ultrasonicator and Illumina sequencing libraries were prepared from 2ng of sheared cDNA using IDT DNAseq library prep reagents on a Beckman Coulter Biomek i7 according to manufacturer’s protocol. The finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 2200. Uniquely dual indexed libraries were pooled in equimolar ratios and evaluated for cluster efficiency and pool balance with shallow sequencing on an Illumina MiSeq. Final sequencing was performed on an Illumina NovaSeq X Plus with paired-end 150bp 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.