What applications is the Watchmaker RNA Library Prep Kit with Polaris suitable for?
The Watchmaker RNA Library Prep Kit with Polaris workflow is ideally suited for:
- Whole transcriptome sequencing, where both protein coding and noncoding transcripts are interrogated, from both high-quality and degraded samples
- Gene expression analysis
- Isoform/splice variant/gene fusion identification
- Single nucleotide variant detection
- Novel transcript discovery
- Targeted sequencing protocols employing hybridization capture
Which species are the Watchmaker RNA Library Prep Kits with Polaris compatible with?
The Watchmaker RNA Library Prep Kit with Polaris is compatible with depletion of rRNA in human, mouse and rat as well as globin from human samples. It can also deplete rRNA from species similar to human, mouse and rat depending on the sequence similarity of the rRNA.
What targets does the Watchmaker RNA Library Prep Kit with Polaris target for depletion?
- 28S, 18S, 5.8S, and 5S cytoplasmic rRNAs
- 16S and 12S mitochondrial rRNAs
- 45S ETS and ITS rRNAs (probes designed for human only)
- HBA1/2, HBB, HBD, HBM, HBG1/2, HBE1, HBQ1, and HBZ globin RNAs (probes designed for human only)
Why use rRNA and globin depletion for enrichment of RNA?
The use of the Watchmaker RNA Library Prep Kit with Polaris workflow provides depletion of rRNA and globin for human, mouse and rat (HMR). Polaris depletion allows for a more accurate representation of the whole transcriptome, just excluding rRNA and globin transcripts. This workflow retains intronic and intergenic regions, which is where many long non-coding transcripts are found. It is also compatible with degraded inputs due to enzyme-based depletion mechanisms.
In comparison, a poly(A) capture workflow is useful when specifically interrogating mRNA species. The poly(A) capture approach is incompatible for use with degraded RNA, where there is the possibility of strand breaks between the 3′ polyadenylated region and the rest of the transcript.
What is the starting material I need to use?
This kit is compatible with both high- and low-quality samples, including FFPE and blood-derived, suspended in 18 µL of RNase-free water.
For input into Polaris depletion high-quality total RNA ranging from 1 – 1000 ng has been tested and shown to produce high-performing libraries. While libraries generated from FFPE samples can be more dependent on FFPE quality, libraries have been successfully generated with input amounts as low as 10 ng with FFPE-derived RNA.
For inputs lower than 1 ng, please contact email@example.com
What are the RNA purity requirements?
RNA inputs should be free from contaminating DNA. If the total RNA contains DNA, remove the contamination by incubating with DNase I (not supplied with kit). RNA should be suspended in RNase-free water and be free of salts (e.g., Mg2+, or guanidinium salts), chelating agents (e.g., EDTA or EGTA), and organics (e.g., phenol or ethanol).
Why must the RNA be free of DNA?
Any DNA present in an RNA sample can be carried through to the final library. This can cause data distortion, especially with genes with low expression and any non-coding RNA quantification.
Do you have specific recommendations for using FFPE with the Watchmaker RNA Library Prep Kit with Polaris?
Yes, we have developed an FFPE-specific workflow, which has been optimized for low quality input material. This includes an FFPE Treatment Step as well as optimal adapter ligation and amplification parameters. Please refer to Library Construction Protocol B: FFPE samples in the User Guide for more information.
What is the purpose of the FFPE treatment step?
Crosslinks that form during sample fixation are not always adequately reversed during RNA extraction. Our FFPE treatment step helps chemically reverse residual crosslinks to make more template available during library preparation and amplification.
Is the FFPE step required?
It is highly recommended for FFPE samples and acts to increase yield and downstream data quality for FFPE samples. It has minimal detrimental effects on downstream data.
I am only interested in coding RNA. Can I use the Polaris Depletion Kit (Human/Mouse/Rat) to remove globin mRNA from previously enriched mRNA?
Yes, please contact firstname.lastname@example.org for recommendations for workflow optimization depending on your mRNA capture of choice.
Which adapters are the Watchmaker RNA Library Prep Kit with Polaris compatible with?
This kit is compatible with adapters that have a 3′ overhanging T to facilitate adapter orientation during dscDNA ligation. Note that adapter quality impacts overall library preparation efficiency. Ensure that adapters are adequately duplexed and at the appropriate concentration prior to use. For a list of commercially available adapters and more detailed adapter recommendations, please contact email@example.com.
Is vortex mixing safe for reactions containing RNA and/or enzyme?
Vortex mixing is recommended for master mix generation and subsequent addition to sample. Pipette mixing is an acceptable alternative so long as care is taken to ensure a completely homogeneous reaction.
What is the shelf life and recommended storage conditions?
The Watchmaker RNA Library Prep Kit with Polaris has a shelf life of 12 months upon receipt, provided it is stored at -20॰C ±5॰C and handled under recommended storage conditions.