Question: Can I use RNase-rich samples for single cell gene expression or Multiome assays?
Answer: RNases are molecules that protect organisms from pathogens by indiscriminately digesting RNA. As a result, RNase activity poses significant challenges in capturing high quality mRNA and subsequently generating high quality gene expression data. Additional considerations and protocol modifications, including those discussed below, may be necessary for RNase-rich tissues to maintain complexity in final gene expression libraries.
During sample processing workflow, RNases can be released that contribute to RNA degradation. Optimization will be necessary when working with RNase-rich samples. Below are some key considerations when working with RNase-rich samples.
1. Maintain an RNase free environment
When working with RNA, a general recommendation is to maintain an RNase-free environment by regularly cleaning surfaces and pipettes using an RNase decontaminating solution (i.e. RNase Away, RNase Zap). Utilize RNAse-free tubes, pipettes, and reagents to avoid further contamination of your samples. General guidelines for maintaining an RNase free lab can be found here: https://bitesizebio.com/163/10-ways-to-work-rnase-free/.
2. Shorten sample processing time and maintain samples at 4C
Long sample processing times give RNases a chance to degrade RNA. Minimize sample processing steps, keep samples on ice throughout the workflow whenever possible, and avoid long enzymatic incubations at 37°C (optimal temperatures for RNases).
3. Use RNase inhibitor
RNase inhibitors are recommended for preparation of RNase-rich tissue. RNase inhibitors can be added to cell or nuclei resuspension buffers and nuclei isolation buffers. Up to 1U/ul may be used for single cell gene expression assays. See: Are RNase inhibitors required in the preparation of my sample?.
For the Multiome ATAC+GEX assay, adding 1U/ul Protector RNase inhibitor in all buffers is critical. See: Can I use an alternative RNase inhibitor part number?.
4. Process GEMs quickly
After transferring the GEMs from the chip, they can be safely placed on ice for up to 1 h. However, with RNase-rich samples, we recommend proceeding to GEM incubation as soon as possible with minimal delay.
5. Increase cDNA amplification cycles by 1-2 cycles
Due to high RNase activity, some samples may benefit from additional cDNA amplification cycles to obtain sufficient cDNA for library generation. Always QC the resulting cDNA to make sure its profile is as expected. See: What is the expected size range for amplified cDNA?
6. Nuclei isolation may be more challenging than cell isolation for RNase rich tissues
Most cells remain intact when isolating cells from tissue, and fewer RNases leak out. When isolating nuclei, cell membranes must be lysed resulting in release of cytoplasmic contents including RNases. Although RNase inhibitor is added, this may not be enough to counteract all RNases present in RNase-rich tissues. We have performed very limited testing of RNAse rich tissues on gene expression products (3', 5', Multiome ATAC+GEX). Users have reported challenges in obtaining sufficient cDNA yield from RNase-rich sample types (e.g. splenocytes). Caution is recommended for nuclei isolation from RNase-rich samples. If possible, use whole cells when possible for 3' gene expression assays. If nuclei must be isolated, consider a pilot experiment to optimize sample preparation before continuing with additional samples. See: What metrics should I be looking at to optimize my sample prep in my pilot experiment?
|RNase rich tissues||Citation||Relevant 10x Publications|
|https://pubmed.ncbi.nlm.nih.gov/556267/||List available here|
|Spleen||https://www.jbc.org/article/S0021-9258(18)34185-1/pdf||List available here|
|Lung||https://www.frontiersin.org/articles/10.3389/fimmu.2019.02626/full||List available here|
Products: Single Cell Immune Profiling, Single Cell Gene Expression, Single Cell Multiome ATAC + GEX