Using the NEXTFLEX Small RNA-Seq Kit v3 for alternative RNA inputs, including RIP-Seq, CLIP-Seq, HITS-CLIP, and Ribosomal Profiling
Although NEXTFLEX Small RNA Sequencing Kits are normally used to prepare sequencing libraries representing miRNAs, siRNAs, or piRNAs, the kits can also be used to create libraries from other RNA samples, such as RNA isolated from ribosomal profiling, RIP-Seq (RNA binding protein immunoprecipitation and sequencing), CLIP-Seq (cross-linking immunoprecipitation and sequencing), and HITS-CLIP experiments. To create sequencing libraries with NEXTflex Small RNA Sequencing Kits, the RNA of interest should have a 5′ monophosphate and 3′ hydroxyl. Fortunately, these modifications can easily be added to most RNA molecules with T4 polynucleotide kinase (PNK). PNK contains both 5′ kinase and 3′ phosphatase activities, making it ideal for preparing RNA samples for library preparation with the NEXTFLEX Small RNA Seq Kit v3.
In order to prepare a library from RNA molecules that do not already contain a 5′ monophosphate and 3′ hydroxyl, the following basic strategy can be followed:
- Treat RNA with T4 PNK according to the manufacturer’s instructions. A 10 minute pre-incubation with all components except ATP may help increase the phosphatase activity, which will lead to greater yield for RNA samples that contain 3′ phosphates, such as those that have undergone chemical/heat fragmentation.
- Purify RNA using ethanol precipitation or a column-based kit, such as the Zymo® RNA Clean and Concentrator-5. Resuspend/elute the RNA in ~12 µL of water.
- Optional Check RNA size and approximate quantity by Agilent® Bioanalyzer®. Note that Bioanalyzer® estimates of concentration are often inaccurate, so this value should be treated as an estimate.
- Prepare libraries using the NEXTFLEX Small RNA-Seq Kit v3. If ≥ 1 ng of PNK-treated RNA is used for library prep, 15 cycles or fewer of PCR should be sufficient.
The default protocol enriches for final library products representing RNA molecules of ~20-30 nt. In order to retain products representing larger RNA fragments, the protocol should be modified according to the instructions in the NEXTFLEX Small RNA Seq Kit v3 No Size Selection Supplement.
- If a final size selection is desired, gel-based size selection can be performed, or the volumes used in Step H1: Gel-Free Size Selection & Cleanup can be optimized for the desired size range. Note that bead-based methods only achieve coarse size selection, so PAGE purification is recommended if precise size selection is needed.
What is the lowest input of total RNA possible with this kit?
The lowest recommended input is 1 ng of total RNA. This amount of RNA should work well for most sample types. For samples that have high miRNA content, less total RNA may be used.
Can the Sage® Pippin Prep® system be used for automated size selection of small RNA libraries created with the NEXTFLEX Small RNA Sequencing Kit v3?
Yes, a protocol is available for automated size selection of small RNA libraries constructed using the NEXTFLEX Small RNA Sequencing Kit v3. Download the Sage® Pippin Prep® protocol for the NEXTFLEX Small RNA-Seq Kit v3.
Why do I have to perform a cleanup after 3′ ligation? This step is not necessary in other kits.
Typical “tricks” to reduce formation of adapter-dimer do not work well when using adapters with randomized ends, which is the reason for the Excess 3′ Adapter Depletion step.
Is this kit compatible with both single read and paired-end sequencing?
Yes, in addition to single read sequencing this kit can also be used with paired-end sequencing.
Will the random nucleotides from the adapters be present in my sequencing reads?
Yes, the random bases will be present as the first four bases of the read and the four bases immediately before the 3′ adapter sequence.
How should the random bases be handled for alignment?
Following 3′ adapter trimming, the first and last four bases of the read should be trimmed prior to alignment. Another option is to use an aligner with a “local” mode.
What sequencing platforms is this kit compatible with?
This kit is compatible with all common Illumina® sequencing platforms including the HiSeq®, MiSeq®, and NextSeq® 500 systems.
How many sequencing cycles do small RNA libraries need?
This is dependent on your experiment and whether you’re looking at microRNAs or long non-coding RNAs. For microRNAs, we do not recommend fewer than 36 sequencing cycles. The Illumina® MiSeq® 1×50 cycle run is most commonly used for sequencing microRNAs. Most microRNAs are between 15–30 bases long, and sequencing beyond this point only sequences the adapter.
How many reads are needed per sample for small RNA sequencing?
This is also dependent on your experiment. Generally for expression profiling, 1–2M mapped reads is an accepted range. For discovery applications, you may want to increase to 2–5M reads. Contact us at [email protected] and we can help you determine the best number of reads based on your experimental design.
How are the barcoded primers incorporated into the small RNA-Seq libraries?
The NEXTFLEX Small RNA Barcodes Primers, each containing a six-base index, are incorporated into the library during the PCR amplification step. This design allows for the indexes to be read using a second read, which reduces bias.
During size selection on 6% PAGE gel, which bands should I cut out of the gel?
MicroRNAs that are between 20 – 30 nt in length will yield a band ~150 -160 bp in length, which should be cut out of the 6% PAGE gel. Do not cut out the ~130 bp band as this is adapter dimer.
What is AIR™ Ligase and why is it used in the NEXTFLEX Small RNA-Seq Kit v3?
AIR Ligase is an enhanced, truncated T4 RNA Ligase, which increases the efficiency with which small RNAs are tagged with adapter, giving greater sequence depth.
This kit recommends up to 25 cycles of PCR amplification for low-input libraries. Won’t this many PCR cycles intro bias?
No, PCR has been shown to introduce negligible bias into small RNA libraries. See publications by Jayaprakash, et al., Hafner, et al. and our whitepaper for more information.
Jayaprakash, A.D., et al., Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing. Nucleic Acids Res, 2011. 39(21): p. e141.
Hafner, M., et al., RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA, 2011. 17(9): p. 1697-712.
What should I do if I observe precipitate in buffers?
Buffers may form precipitate after freezing. If precipitate is seen, bring to room temperature then vortex until the precipitate is in solution. The performance of the buffer is not affected once precipitate is in solution.
What should I do if I observe precipitate in NEXTFLEX® Small RNA PCR Master Mix?
NEXTFLEX® Small RNA PCR Master Mix may a form precipitate after storage at -20°C. Even though this reagent contains an enzyme, it is a hardy enzyme which is resistant to inactivation by vortexing or heating. Therefore, it is safe to bring this reagent to room temperature then briefly vortex until the precipitate is in solution. The performance of this reagent is not affected once the precipitate is in solution.