Bulk RNA Sequencing of Low-Abundance RNA Samples
The Single Cell Genomics Facility (SCGF) specializes in running bulk RNA-Seq projects for samples with a limited amount of total RNA. Our facility offers three different services to process bulk RNA-Seq samples.
Bulk RNA Sequencing Assays
- Ovation RNA-Seq System v2 Assay (Tecan Genomics) + Nextera XT DNA Library Preparation Kit (Illumina)
- Revelo RNA-Seq High Sensitivity Assay (Tecan Genomics)
- SMART-Seq mRNA Assay (Takara) + Nextera XT DNA Library Preparation Kit (Illumina)
The Ovation RNA-Seq System v2 assay amplifies total RNA samples between 500pg – 10ng, to create 2 – 4ug of double stranded cDNA. The Nextera XT DNA Library Preparation Kit is then used to create DNA library templates from the double stranded cDNA. The SCGF recommends using this service if the total quantity of your RNA per sample is less than 100ng and/or if your lab is interested in sequencing both polyadenylated and non-polyadenylated transcripts. To use this assay, the samples should have concentrations greater than 100 pg/uL. The minimum amount of RNA required is 500pg. However, it is recommended to use at least 2ng of total RNA per sample.
Revelo RNA-Seq High Sensitivity Assay
The Revelo RNA-Seq High Sensitivity Assay is a kit optimized for degraded, low-input human or mouse samples. This technology uses 250pg – 10ng of total RNA as starting material. The protocol begins with a DNase treatment (to remove DNA contamination from the RNA) and an optional SPIABoost. SPIABoost is a method used to suppress human rRNA, human globin mRNA, or mouse rRNA allowing transcripts of interest to be focused. Next, priming for first strand synthesis occurs at the 3’ end and throughout the RNA strand. Once complete, second strand synthesis occurs using SPIA (Single Primer Isothermal Amplification) followed by purification and library preparation.
SMART-Seq mRNA Assay
The SCGF uses the SMART-Seq mRNA Assay (Takara) to create full-length cDNA from very low amounts of total RNA. This assay is designed to select and sequence polyadenylated transcripts. Specifically, the SMART-Seq mRNA Kit amplifies RNA samples with less than 50pg, to create 3 – 17ng of cDNA. The Nextera XT DNA Library Preparation Kit is used to create DNA library constructs from the cDNA.
Bulk RNA Sequencing Sample Requirements
- Purified total RNA
- QC check of RNA prior to beginning experiment (performed by SCGF)
Bulk RNA Sequencing Checklist
- Identify bulk RNA sequencing assay to be used.
- Submit RNA samples to SCGF for QC.
- QC requirements:
- Ovation RNA-Seq System v2 Assay: QC RIN score >7
- Revelo RNA-Seq High Sensitivity Assay: QC RIN score >2 or DV200 of 30%
- SMART-Seq mRNA Assay: QC RIN score >7
- Review QC results and decide which samples will proceed into the bulk RNA sequence experiment.
