AQRNA-seq for Quantifying Small RNAs

NOTE: Figure 1 provides a graphical illustration of the procedures involved in AQRNA-seq library preparation. Detailed information regarding the reagents, chemicals, and columns/kits used in the procedure can be found in the Table of Materials. It is recommended to perform a comprehensive evaluation of purity, integrity, and quantity of the input RNA samples using (i) 3% agarose gel electrophoresis, (ii) automated electrophoresis tools for sample quality control of biomolecules (see Table of Materials), and (iii) UV-Visible spectrophotometry and/or fluorometric quantification. It is mandatory to keep all reactions and master mixes on ice, unless otherwise specified. Transport reagents (e.g., enzymes) in cool boxes to and from -20 °C storage to preserve their shelf lives and avoid multiple freeze-thaw cycles of library intermediates.

1. Dephosphorylation of RNAs

NOTE: Removal of the 5'-phosphate (P; donor) prevents self-ligation to the 3'-hydroxyl (OH; acceptor) of RNAs. Linkers will not self-ligate as their 3' end is modified to incorporate either a dideoxycytidine (Linker 1) or a spacer (Linker 2). Linkers can only be ligated by joining their 5'-P to the 3'-OH of the RNAs or cDNAs.

1. Prepare the dephosphorylation reaction in a sterile PCR tube (e.g., 200 µL or 500 µL tube) by adding up to 2 µL of the RNA sample, 0.5 µL of 40 U/µL RNase inhibitor, 1 µL of 0.5 µM internal standard (Table 1), 0.5 µL of 10x T4 RNA ligase reaction buffer, 1 µL of 1 U/µL Shrimp alkaline phosphatase, and sufficient RNase-free water to bring the overall volume to 5 µL.
   NOTE: For typical samples, 75 ng of RNAs (or approximately 2 pmol for an 80 nt RNA) is considered sufficient for quantification of small RNAs using this protocol.
2. Incubate at 37 °C for 30 min to dephosphorylate RNAs and then at 65 °C for 5 min to inactivate the enzyme and denature the RNAs. Keep samples at 4 °C for at least 10 min to prevent renaturation.

2. Ligation of Linker 1 to the 3' end of RNAs

1. Prepare the Linker 1 ligation reaction in a sterile PCR tube by adding 5 µL of the dephosphorylated RNAs (products from step 1), 0.5 µL of 40 U/µL RNase inhibitor, 1 µL of 100 µM Linker 1 (Table 1), 3 µL of 10 mM ATP, 2.5 µL of 10x T4 RNA ligase reaction buffer, 15 µL of PEG8000 (50% solution), 2 µL of 30 U/µL T4 RNA ligase 1, and 1 µL of RNase-free water.
   NOTE: Reagents can be made into master mix to facilitate processing the samples. Do not include PEG8000 and T4 RNA ligase 1 in the master mix.
2. Incubate at 25 °C for 2 h and then at 16 °C for 16 h to ligate Linker 1 to the RNAs.
3. Column-purify the Linker-1-ligated RNAs. Use a kit for DNA/RNA recovery and clean-up (see Table of Materials).
   NOTE: This column purification protocol applies to all subsequent column purification using the same kit. Kits that use gel-filtration technology to remove dye terminators from sequencing reactions (see Table of Materials) cannot be used here, as PEG8000 is incompatible with the gel filters of such kits. It is recommended to save an aliquot (1.2 µL) of each sample after purification. When necessary, these aliquots can be used for checking the ligation efficiency by running a commercial nucleic acid analyzer. Keep the remaining purified samples on ice or at -20 °C until further steps.
   1. For sample volume < 50 µL, add RNase-free water to bring it to 50 µL. Add 2 volumes of the oligo binding buffer (provided in the kit) to 1 volume of the sample. Add 8 volumes of 100% ethanol to 1 volume of the sample.
   2. Load the sample (up to 750 µL at a time) to a column placed inside a 2 mL collection tube (provided in the kit). To bind RNAs to the column, centrifuge at 10,000 x g for 30 s and discard the flow-through (i.e., the liquid in the collection tube). Place the column back into the collection tube.
   3. To wash the impurities off the column, add 750 µL of the DNA wash buffer (provided in the kit) to the column. Centrifuge at 10,000 x g for 30 s and discard the flow-through. Place the column back into the collection tube.
   4. Centrifuge at maximum speed (e.g., 16,000 x g for a benchtop microcentrifuge) for an additional 1 min to remove residual DNA wash buffer.
   5. To elute the RNAs, carefully move the column to a sterile 1.5 mL tube, add RNase-free water to the column. Use a greater volume than needed when eluting the RNAs to account for potential volume loss during the elution process. For instance, if 15 µL is required for the following step, add 17 µL of RNase-free water for elution. Centrifuge at 10,000 x g for 30 s.

3. Removal of post-transcriptional methylations by AlkB demethylase

NOTE: AlkB is a bacterial enzyme that removes methyl groups from some, but not all, methylated nucleotides in DNA and RNA. Removal of several types of methylated ribonucleotides in RNA prevents fall-off of the reverse transcriptase to allow more full-length reads and identification of modification sites. This step needs to be controlled at a low pH to prevent unexpected degradation of RNAs.

1. Prepare a stock solution of 1 M 2-ketoglutarate by dissolving 1.4611 g of 2-ketoglutarate (146.11 g per M) in 10 mL of RNase-free water. Filter sterilize the solution through a 0.2 µm syringe filter. Aliquot the stock solution into 2 mL sterile tubes and store at -20 °C.
2. Prepare a stock solution of 0.5 M L-ascorbic acid by dissolving 0.88 g of L-ascorbic acid (176.12 g per M) in 10 mL of RNase-free water. Filter sterilize the solution through a 0.2 µm syringe filter. Aliquot the stock solution into 2 mL sterile tubes and store at -20 °C.
3. Prepare a stock solution of 0.25 M ammonium ferrous sulfate hexahydrate by dissolving 0.9835 g of ammonium ferrous sulfate hexahydrate (392.14 g per M) in 10 mL of RNase-free water. Filter sterilize the solution through a 0.2 µm syringe filter. Aliquot the stock solution into 2 mL sterile tubes and store at -20 °C.
4. Prepare a stock solution of 1 M HEPES by dissolving 2.383 g of HEPES (238.30 g per M) in 10 mL of RNase-free water. Adjust the pH of the solution to 8 using NaOH and filter sterilize the solution through a 0.2 µm syringe filter. Aliquot the stock solution into 2 mL sterile tubes and store at -20 °C.
5. Prepare a 2x AlkB reaction buffer. To make 10 mL of the buffer, combine 1.5 µL of 1 M 2-ketoglutarate (made in step 3.1), 80 µL of 0.5 M L-ascorbic acid (made in step 3.2), 6 µL of 0.25 M ammonium ferrous sulfate hexahydrate (made in step 3.3), 100 µL of 10 mg/mL BSA, 1000 µL of 1 M HEPES (made in step 3.4; add last), and 8812.5 µL of RNase-free water. Filter sterilize the buffer through a 0.2 µm syringe filter.
   NOTE: The 2x AlkB reaction buffer must be prepared fresh immediately prior to each experiment due to the chemical lability of the components.
6. Prepare the AlkB digestion reaction in a sterile PCR tube by adding 20 µL of the Linker-1-ligated RNAs (products from step 2), 50 µL of the 2x AlkB reaction buffer (made in step 3.5), 2 µL of AlkB demethylase, 1 µL of RNase inhibitor, and 27 µL of RNase-free water.
7. Incubate at room temperature for 2 h to remove post-transcriptional methylations from the RNAs.
8. To remove AlkB from the reaction, follow the steps described below.
   1. For a clean phase separation, add 50 µL of RNase-free water into the AlkB reaction, and then add 100 µL phenol: chloroform: isoamyl alcohol 25:24:1 (pH = 5.2).
   2. Shake by hand for 10 s, and then centrifuge at 16,000 x g for 10 min. Make sure that the rotor of the benchtop centrifuge is compatible with the PCR tubes. Use adaptors if needed.
   3. Transfer the RNAs (i.e., the aqueous layer on top; approximately 140 µL) into a sterile 1.5 mL tube. If chloroform (i.e., the bottom layer) is mixed into the aqueous layer, centrifuge again with the same settings.
   4. Add 100 µL of chloroform to the extracted RNAs to remove residual phenol. Shake by hand for 10 s, and then centrifuge at 16,000 x g for 10 min.
   5. Transfer the RNAs (i.e., the aqueous layer on top; approximately 120 µL) into a sterile 1.5 mL tube.
9. Column-purify the extracted RNAs. Use a kit for DNA/RNA recovery and clean-up (see Table of Materials). Follow the protocol detailed in step 2.3 to perform the column purification.

4. Removal of excess Linker 1

NOTE: It is recommended to save an aliquot (1.2 µL) of each sample after purification. When necessary, these aliquots can be used for checking the efficiency of RecJf digestion by running a commercial nucleic acid analyzer. Proceed with the purified samples immediately to reverse transcription.

1. Prepare the deadenylation reaction in a sterile PCR tube by adding 15 µL of Linker-1-ligated RNAs (products from step 3), 1 µL of 40 U/µL RNase inhibitor, 2 µL of 10x kit buffer 2 (see Table of Materials), and 2 µL of 50 U/µL 5'-deadenylase.
2. Incubate at 30 °C for 1 h to remove the adenine at the 5' end of Linker 1. Add 2 µL of 30 U/μL RecJf into the deadenylation reaction.
3. Incubate at 37 °C for 30 min to digest excess Linker 1. Add another 2 µL of 30 U/μL RecJf into the reaction.
4. Incubate at 37 °C for 30 min to continue the digestion of excess Linker 1 and then at 65 °C for 20 min to denature the enzyme.
5. Column-purify the Linker-1-ligated RNAs. Use a kit that uses gel-filtration technology to remove dye terminators from sequencing reactions (see Table of Materials), as it is effective at removing short remnants (e.g., oligonucleotides with a length of 2 to 10 bp). Follow the steps described below for purification.
   1. Prepare gel-filter columns according to the manufacturer's protocol. Place a column into a sterile 1.5 mL tube and load the column with 24 µL of the sample.
   2. To purify the RNAs, centrifuge at 800 x g for 3 min and discard the column. The purified RNAs are in the eluent.

5. Reverse transcription (RT) reaction

NOTE: The following RT (see Table of Materials) reaction setup follows the manufacturer's protocol, with minor modifications to allow for AQRNA-seq compatibility.

1. Prepare the RT primer annealing reaction in a sterile PCR tube by adding 24 µL of template RNAs (products from step 4), 1 µL of 2 µM RT primer (Table 1), and 1 µL of dNTP (10 mM of each type of nucleotides).
2. Incubate at 80 °C for 2 min to anneal RT primers to the template RNAs, and then cool on ice immediately for 2 min.
3. Prepare the RT reaction by adding 6 µL of 5x RT reaction buffer, 1 µL of 40 U/µL RNase inhibitor, and 1 µL of reverse transcriptase into the annealing reaction tube.
4. Incubate at 50 °C for 2 h to reverse transcribe the RNA templates, and then at 70 °C for 15 min to inactivate the enzyme. The RT products (i.e., RNA-cDNA hybrids) can be stored at 4 °C or -20 °C overnight.

6. RNA hydrolysis

1. Add 1 µL of 5 M NaOH into the RNA-cDNA hybrid (products from step 5). Incubate at 93 °C for 3 min to hydrolyze the RNA strand of the RNA-cDNA hybrid.
2. Add 0.77 µL of 5 M HCl to neutralize the reaction. After adding HCl, flick to mix, and spin down the tube. Neutralization is instantaneous.
   NOTE: It is recommended to test the precise amount of 5 M HCl that is needed for neutralizing 1 µL of NaOH (e.g., using pH strips) in buffered conditions.
3. Column-purify the single-stranded cDNAs. Use a kit for DNA/RNA recovery and clean-up (see Table of Materials). Follow the protocol detailed in step 2.3 to perform the column purification.
   NOTE: Kits that use gel-filtration technology to remove dye terminators from sequencing reactions (see Table of Materials) cannot be used here due to the pH variation in previous steps.
4. Speed-vac the purified cDNAs to < 5 µL and then add RNase-free water to bring the volume back to 5 µL. Be careful not to speed-vac the cDNAs to complete dryness.
5. Transfer the purified cDNAs into a sterile PCR tube. The purified cDNAs can be stored at -20 °C for up to 1 week.

7. Ligation of Linker 2 to the 3' end of cDNAs

1. Prepare the Linker 2 ligation reaction in a sterile PCR tube by adding 5 µL of cDNAs (products from step 6), 1 µL of 50 µM Linker 2 (Table 1), 2 µL of 10x T4 DNA ligase reaction buffer, 1 µL of 10 mM ATP, 9 µL of PEG8000 (50% solution), and 2 µL of 400 U/µL T4 DNA ligase.
   NOTE: Reagents can be made into master mix to facilitate the processing of the samples. Do not include PEG8000 and T4 DNA ligase in the master mix.
2. Incubate at 16 °C for 16 h to ligate Linker 2 to the cDNAs.
3. Column-purify the Linker-2-ligated cDNAs. Use a kit for DNA/RNA recovery and clean-up (see Table of Materials). Follow the protocol detailed in step 2.3 to perform the column purification.

8. Removal of excess Linker 2

1. Prepare the deadenylation reaction in a sterile PCR tube by adding 16 µL of Linker-2-ligated cDNAs, 2 µL of 10x kit buffer 2, and 2 µL of 50 U/µL 5'-deadenylase. Incubate at 30 °C for 1 h to remove the adenine at the 5' end of Linker 2.
2. Add 2 µL of 30 U/μL RecJf into the deadenylation reaction. Incubate at 37 °C for 30 min to digest excess Linker 2. Add another 2 µL of 30 U/μL RecJf into the reaction.
3. Incubate at 37 °C for 30 min to continue the digestion of excess Linker 2 and then at 65 °C for 20 min to denature the enzyme.

9. PCR amplification of the cDNAs with sequencing primers

1. Assign PCR primers to the samples. Each sample needs a unique combination of forward and reverse primers (Table 1) for effective multiplexing.
2. Add RNase-free water to bring the sample volume to 25 µL. Save 5 µL of each sample into a sterile PCR tube as a backup in case PCR needs to be repeated.
3. Prepare the PCR reaction (see Table of Materials for PCR kit) by adding 20 µL of cDNAs (products from step 8), 1 µL of 2.5 µM forward primer, 1 µL of 2.5 µM reverse primer, 25 µL of 2x DNA polymerase buffer, 2 µL of RNase-free water, and 1 µL of DNA polymerase.
   NOTE: Reagents can be made into master mix to facilitate the processing of the samples. Do not add DNA polymerase to the master mix.
4. Perform PCR with an initial denaturation at 94 °C for 1 min, followed by 18 cycles of denaturation at 98 °C for 20 s – annealing at 58 °C for 20 s – extension at 68 °C for 1 min.
   NOTE: Do not PCR amplify beyond the linear range. A total of 18 cycles will be optimal for most experiments, but this may be context dependent.
5. Speed-vac the PCR products to less than 25 µL and then add RNase-free water to bring the volume back to 25 µL. Transfer 5 µL of the PCR products to a sterile 0.5 mL tube for checking the size distribution (see step 9.6). Store the remaining 20 µL of the PCR products at -20 °C until further steps.
6. Check the size distribution of the PCR products as described below.
   1. Prepare 3% agarose gel in TAE buffer.
      NOTE: In this protocol, ethidium bromide (EtBr) is used for post-electrophoresis gel staining (see step 9.6.5). Appropriate DNA stains can be added into the gel solution at this step or used for gel staining later.
   2. Mix 1 µL of 6x loading dye into 5 µL of PCR products (from step 9.5) and load the gel with the mixture.
   3. Load 5 µL of DNA ladders into the well before the first sample and the well after the last sample. Use 50 bp or 100 bp DNA ladders to allow for improved size discrimination of PCR products between 150 bp and 300 bp.
   4. Run gel electrophoresis to locate the PCR products. The appropriate running condition may be context dependent. Here, run at 120 V, 400 mA for 75 min for a 17.78 cm (width) x 10.16 cm (height) x 1 cm (thickness) gel slab.
   5. Place the gel in a box and fill the box with deionized (DI) water until the gel is completely immersed. Add 10 µL of EtBr into the DI water soaking the gel. Wrap the box with foil and place it on a shaker. Stain the gel for 30 min while shaking.
   6. Discard the EtBr-containing waste into a waste bottle placed in a fume hood. Rinse the gel with DI water once and discard the EtBr-containing water into the waste bottle.
   7. Fill the box with DI water until the gel is completely immersed. Wrap the box with foil and place it on a shaker. Wash the gel for 10 min while shaking.
   8. Discard the EtBr-containing water into the waste bottle in the fume hood. Use a gel imager to visualize the bands. Acquire a high-resolution image of the gel.

10. Gel purification

1. Prepare 3% agarose gel in TAE buffer. Make a 1 cm thickness gel with wide combs (1 mm thickness; 5 mm width; 15 mm depth), such that each well can contain at least 25 µL of sample-loading dye mixture.
2. Mix 4 µL of 6x loading dye with 20 µL of PCR products (from step 9.5) and load the gel with the mixture. Leave empty lanes in between samples to minimize cross-contamination during gel excision.
3. Load DNA ladders, run gel electrophoresis, stain and wash the gel, and take gel images as described in step 9.6.
4. Excise the gel blocks that contain PCR products within the target size range. To minimize contamination with primer dimers (175 bp linkers with no inserts), extract PCR products with size above 195 bp (175 bp linkers + 20 bp miRNAs).
5. Purify the PCR products using gel extraction. Use a gel extraction kit (see Table of Materials). The purification protocol is based on the manufacturer's protocol, with minor modifications for AQRNA-seq compatibility. All centrifugation steps should be conducted at 17,900 x g for 1 min using a benchtop centrifuge at room temperature, unless otherwise specified. Follow the steps described below.
   1. Measure the weight of the gel blocks inside the tubes. Add 6 volumes of Buffer QG (provided in the kit) to 1 volume of gel block (1 mg gel is approximately 1 µL).
   2. Incubate at 50 °C for 10 min or until complete dissolution of the gel blocks. Vortex tubes every 2 min to facilitate gel dissolution. After dissolving the gel, the mixture should resemble the color of the Buffer QG without the dissolved gel. If the color is orange or violet, add 10 µL of 3 M sodium acetate (pH = 5.0) and mix thoroughly.
   3. Add 1 gel volume of isopropanol to the mixture and mix thoroughly. Place a spin column in a 2 mL collection tube (provided in the kit).
   4. To bind DNA, apply the sample (up to 750 µL each time) to the column and centrifuge. Discard the flow-through and place the column back into the same collection tube. The maximum amount of gel per spin column is 400 mg.
   5. Add 500 µL of Buffer QG to the column and centrifuge. Discard the flow-through and place the column back into the same collection tube.
   6. To wash the impurities, add 750 µL of Buffer PE (provided in the kit) to the column, let the column stand for 5 min, and centrifuge. Discard the flow-through and place the column back into the same collection tube. Centrifuge once again to remove residual wash buffer.
   7. Place the column into a sterile 1.5 mL tube. To elute DNA, add 30 µL of Buffer EB (provided in the kit) to the center of the column membrane, let the column stand for 4 min, and centrifuge.
   8. Speed-vac and resuspend the gel-purified PCR products in 12 µL of Buffer EB.
6. Measure the concentration of the constructed libraries by UV-Visible spectrophotometry and/or fluorometric quantification.

11. Library sequencing

1. Submit the constructed libraries to an external sequencing center for quality assessment and Illumina sequencing. To ensure sufficient sensitivity in quantitative mapping of small RNA landscapes, opt for paired-end sequencing with 75-bp reads from each direction (i.e., PE75), aiming for at least 1.5 M raw sequence reads in each direction for each sample. Use custom primers (Table 1) for NextSeq sequencing, but this is optional for sequencing on MiSeq.
   NOTE: Sequencing can be performed using MiSeq or NextSeq500 platforms. The choice of platform may depend on the nature of samples and the total sample count.

12. Data analytics pipeline

NOTE: Figure 2 provides a graphical illustration of simplified procedures involved in the data analytics pipeline, which takes raw sequence reads (in FASTQ format) as the input and generates an abundance matrix with rows representing members of small RNA species of interest and columns representing samples. For paired-end sequencing, each sample corresponds to two FASTQ files, one for the forward reads and the other for the reverse reads. The complete data analytics pipeline, with all the associated scripts and a manual with extensive annotations for each step, is available at GitHub (https://github.com/Chenrx9293/AQRNA-seq-JoVE.git).

1. Retrieve raw sequence reads from the external sequencing center and assess sequencing quality using open-source programs such as FastQC¹⁴ or fastp¹⁵.
2. Create a reference sequence library in FASTA format.
   NOTE: The key to the pipeline's adaptability to diverse small RNA classes is an appropriate reference sequence library. To achieve accurate abundance estimates of members of specific RNA classes of interest (e.g., miRNA), the users are anticipated to meticulously curate a reference sequence library for use with the pipeline. All the other instructions for pipeline execution remain consistent across different small RNA classes.
3. Create a directory named AQRNA-seq for implementing the data analytics pipeline and place all scripts, quality-filtered sequence reads, and the reference sequence library into this directory. Follow the detailed instructions on GitHub (https://github.com/Chenrx9293/AQRNA-seq-JoVE.git) to prepare sub-directories and make essential modifications to the files for targeting different organisms and/or small RNA species, as well as compatibility of the operating system and/or job scheduler.
4. Implement the data analytics pipeline following the steps outlined in the manual on GitHub (https://github.com/Chenrx9293/AQRNA-seq-JoVE.git), which contains descriptions, input and output files, as well as command lines for each step. In summary, the pipeline encompasses (i) trimming linker sequences and random nucleotides from the reads, (ii) filtering reads based on their length, (iii) mapping the reads to the reference sequences, (iv) resolving ambiguous mappings, and (v) generating the abundance matrix.