Small RNA populations revealed by blocking rRNA fragments in Drosophila melanogaster reproductive tissues

RNA interference (RNAi) is a complex and highly conserved regulatory mechanism mediated via small RNAs (sRNAs). Recent technical advances in high throughput sequencing have enabled an increasingly detailed analysis of sRNA abundances and profiles in specific body parts and tissues. This enables investigations of the localized roles of microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, variation in the proportions of non-coding RNAs in the samples being compared can hinder these analyses. Specific tissues may vary significantly in the proportions of fragments of longer non-coding RNAs (such as ribosomal RNA or transfer RNA) present, potentially reflecting tissue-specific differences in biological functions. For example, in Drosophila, some tissues contain a highly abundant 30nt rRNA fragment (the 2S rRNA) as well as abundant 5’ and 3’ terminal rRNA fragments. These can pose difficulties for the construction of sRNA libraries as they can swamp the sequencing space and obscure sRNA abundances. Here we addressed this problem and present a modified “rRNA blocking” protocol for the construction of high-definition (HD) adapter sRNA libraries, in D. melanogaster reproductive tissues. The results showed that 2S rRNAs targeted by blocking oligos were reduced from >80% to < 0.01% total reads. In addition, the use of multiple rRNA blocking oligos to bind the most abundant rRNA fragments allowed us to reveal the underlying sRNA populations at increased resolution. Side-by-side comparisons of sequencing libraries of blocked and non-blocked samples revealed that rRNA blocking did not change the miRNA populations present, but instead enhanced their abundances. We suggest that this rRNA blocking procedure offers the potential to improve the in-depth analysis of differentially expressed sRNAs within and across different tissues.

The entire product of this reaction is to be used in the next 5' adapter ligation step.

(iv) 5' adapter ligation
Use a total of 20 pmol of 5' adapter per reaction.
Denature the 5' adapter by heating at 70°C for 2 minutes, then place on ice.
Entire RNA sample following the previous degradase reaction

(vii) PCR Amplification, round 1
Use 4 µl of the cDNA to run a 20 µl PCR reaction.

Note: for each different sample, use a unique index primer
For each sample, run 3 different PCR cycle numbers in order to optimise the reaction. As a guide, use 5, 7 and 9 cycles for the first attempt (if there is no contaminating 30mer band, the cycle number can be increased). • Mix 20 µl PCR product with 5 µl 5x Novex loading dye.
• Load a total of 20 µl in each well, and then mix what is left over from each tube and load that in a fourth lane.
• Load 10 µl of 20 bp ladder (Jena Bioscience) either side of the sample lanes.
• Run the gel in 0.5x TBE buffer for 2-2.5 hours at 120V • Stain the gel with SYBR gold (5 µl in ~50 ml of 0.5x TBE) and scan.
• Print the gel images off in real-size.

(ix) Gel Extraction
• Prepare 0.5 ml tubes by punching 4 holes in the bottom of each with a 21 gauge needle. Put each 0.5 ml tube inside a 2 ml "collection" tube. Use one tube for each gel (or sample).
• Lay the gels over the real-size print-outs and use a razor blade to cut out the area containing the band of interest (re-scan the gels afterwards to check the correct area has been excised).
• Put the slice from each gel into a prepared 0.5 ml tube. Spin the tubes at max speed for 3 minutes to shred the gel slice.
• Discard the 0.5 ml tube. Add 400 µl NEB2 buffer to the broken gel and incubate overnight, shaking at 4ºC.
• Following the overnight incubation, transfer the gel mixture to a Spin-X column (0.45 µm, ThermoFisher), and spin at 2800 RPM for 3 mins, to remove gel debris.
• To 400 µl eluate, add 2 µl glycogen, 40 µl of 3M sodium acetate, and 1200 µl 100% ethanol • Repeat the gel extraction as detailed above. • Once the gel has run, gel extract across all 8 lanes using the protocol above, and resuspend the pellet in final volume of ~10-12 µl

(xii) Adenylation of the 3' HD adapter (if necessary)
Note: Also check that the 3' HD adapter stock is already phosphorylated. • To check for successful 3' adenylation, run adenylated 3' adapter on a PAGE gel (16% urea) against non-adenylated 3' adapter (the stock) and a Scriptminer control.
• Use ~1 pmol of adenylated and non-adenylated adapter. Also mix 1 pmol each of adenylated and non-adenylated together and run this between the other two samples.
• Stain the gel with SYBR gold and view using a scanner • The adenylated adapter should be slightly larger than the non-adenylated. You should see two distinct bands in the mixed sample. The Scriptminer band should be smaller than the others.