Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Small RNA regulate many biological processes. It is therefore important to develop sensitive and unbiased methods to detect them. Our protocol protocol provides steps forward towards this goal.

Our protocol suffers from fewer bias issues, than classical small RNA library appropriation methods, and importantly it allows a more sensitive detections of two prong or meso-RNAs. Such as plant micro-RNAs. After extracting total RNA according to standard protocols.

Pre run a 15%TBE urea gel for fifteen minutes at 200 volts. And mix five to twenty micrograms of total RNA, in a five to fifteen microliter volume, with an equal volume of formamide loading dye in a 200 microliter tube. After five minutes at 65 degree Celsius, in a thermocycler with a heated lid, immediate place the tubes on ice and load the latter and sample on the gel with at least one lane between them.

Then run the gel at 200 volts until the bromophenol has migrated about two-thirds of the length of the gel. To load the RNA, first puncture the bottom of a nuclease free 0.5 milliliter micro centrifuge tube, with a 21 gauge needle, and place the punctured tube in a nuclease free round bottom two milliliter micro centrifuge tube. Next, incubate the gel at room temperature with nucleic acid gel stain in water for ten to fifteen minutes, before viewing the gel on a transilluminator.

Cut out the sample RNA between 17 and 29 nucleotide ladder bands, and transfer the gel piece into the punctured tube. Spin down the gel in a micro centrifuge at maximum speed for two minutes, and remove the 0.5 milliliter tube which should now be empty. Add 300 microliters of nuclease free 0.3 molars sodium chloride to the crushed gel, and place the tube on a rotator for at least two hours at room temperature.

At the end of the end of the incubation, transfer the crushed gel pieces suspension to a spin column, and centrifuge for two minutes at maximum speed in the micro centrifuge. For unligated three prime adaptor elimination, thoroughly mix ten microliters nuclease free water with the extracted RNA sample, and add six microliters of three molars sodium acetate with mixing. Add 40 microliters of magnetic purification beads, and 60 microliters of isopropanol to the sample with thorough mixing.

And incubate the suspension for five minutes at room temperature. At the end of the incubation, place the sample onto a magnetic rack, until the solution becomes clear. Remove the clear supernatant, and add 180 microliters of freshly prepared 80%ethanol to the beads.

After 30 seconds, remove the ethanol and briefly spin the tube. Remove any residual liquid that may have collected at the bottom of the tube, and allow the beads to dry for two minutes before re-suspending the sample in 22 microliters of 10 millimolar Tris. After two minutes magnetize the sample until the solution appears clear.

Next, mix 20 microliters of clear supernatant with six microliters of three molar sodium acetate in a new tube, and add 40 microliters of magnetic beads and 60 microliters of 100%isopropanol. After five minutes at room temperature, magnetize the sample until the solution appears clear, before removing the supernatant. Treat the sample with 180 microliters of freshly prepared 80%ethanol for 30 seconds before removing the ethanol spinning down the sample, and removing any residual liquid that has accumulated at the bottom of the tube.

After allowing the beads to dry for two minutes, re-suspend them in 10 microliters of nuclease free water, for two minutes, before magnetizing the sample until the solution becomes clear. Then, transfer nine microliters of supernatant to a new tube and add one microliter of T4 RNA ligase buffer, and one micrcoliter of water to the sample. For gel purification, run five, ten, and twenty microliters of PCR product on a native 6%TBE gel for about one hour.

Incubate the completed gel with nucleic acid gel stain in water for ten to fifteen minutes, and view the gel on a transilluminator to allow extraction of the 150 base pair library band. As it is difficult to isolate the 150 base pair band representing them, these are our library, due to the presence of closely migrating side products you will need to cut the gel very carefully. Then isolate the RNA as just demonstrated.

For raw sequence file modification, download the fastq sequence files generated during the sequencing run, and preform demultiplexing with bcl2fastq2 as necessary. Remove adaptor sequences using cutodapt, and use the command as demonstrated. Use seqtk as indicated to remove the terminal random nucleotides in the sequences reads.

Then use the awk command as indicated, to discard the sequences shorter than 10 nucleotides. To map the trimmed sequences, open miRBase and download the mature fa file. Use the command as indicated to replace U residues with T residues, to yield a complete list of all the micro RNAs in the database, originating from a variety of organisms.

Select the micro RNA sequences of the organism of interest, with the command as indicated, and map the reads to the file using bowtie2, allowing no mismatches. Use the tool to align the sequences reads to the database, requiring that a read maps entirely to a micro RNA of the database, with out any mismatches as indicated. Then use the command to discard the reads that did not align.

After loading increasing amounts of a PCR amplified library onto a gel as demonstrated, the products corresponding to the expected size can be extracted. After elution, the purified library can be checked on a capillary gel electrophoresis chip. In addition to the expected 150 base pair product, and increasing proportion of a 130 base pair species corresponding to adapter dimers, are typically observed as increasing amount of PCR product are loaded.

Similar results are obtained when libraries from a mix of synthetic small RNAs are prepared using reagents from kits, or other supplies. For comparison, previously published results for the same small RNA mix are shown. Here, a comparison for performance of protocol TS5 with the standard TS protocol for the detection of human unmodified and two prime O-methal modified plant micro RNAs is shown.

The detection of two prime O-methal modified piRNAs in human samples was also tested. As observed TS5 preformed significantly better than TS for the detection of two prime O-methal RNAs, but not for unmodified RNAs. Prepare replicate libraries for each sample at different dates.

Usually there is little variation among replicates made simultaneously, or there can be substantial variation among libraries prepared on different dates.