Small RNA Library Preparation for Next-Generation Sequencing Protocol

RNA Isolation

1. Homogenize tissue samples: Grind 100 mg tissue in liquid nitrogen until fine powder is obtained. Mix 1 mL TRI Reagent® solution with the powder in a microcentrifuge tube.
2. Alternatively, homogenize cultured cells or body fluids: Homogenize and lyse the cells directly by adding 1 mL TRI Reagent® solution per 5–10 × 106 cells or per 10 cm2 culture dish area and pipette up and down.
3. Incubate the mixture for 5 min at room temperature to allow complete dissociation of nucleoprotein complexes.
4. Add 200 μL of chloroform and mix vigorously for 15 s. Incubate at room temperature for a further 5 min.
5. Centrifuge the sample at 16,000 × g at 4 °C for 15 min. The sample should separate into a clear aqueous phase on top, a white interphase in the middle, and a pink organic phase at the base of the tube. Transfer the top, aqueous phase into a fresh microcentrifuge tube, taking care not to take any of the interphase or organic phase. Take note of the volume of the transferred aqueous phase.
6. Add 1 volume of isopropanol to the aqueous RNA extract and mix thoroughly. This can be incubated at room temperature for 10 min or at -80 °C for 30 min. The coprecipitation time and temperature may vary with different samples and may require optimization for the organism or tissue type in question.
7. Centrifuge the sample at 10,000 × g at 4 °C for 10 min and remove the supernatant. A white pellet should have formed at the base of the tube—this is the RNA.
8. Wash the pellet with 1 mL of 80% ethanol. Vortex to dislodge the pellet and centrifuge at 7500 × g for 5 min at 4 °C. Repeat this step once.
9. Remove the residual ethanol and air-dry the RNA pellet for 5–10 min by leaving the tube open. Resuspend the RNA in the desired volume of nuclease-free water and store at -80 °C. If having difficulty getting the RNA into solution, the water and RNA sample can be heated to 70 °C for 5 min.
10. Quantify the RNA concentration using a NanoDrop.

sRNA Purification

1. Mix the RNA elute with three volumes of absolute ethanol, 0.1 volume of sodium acetate (pH 5.0), and 1 μL of glycogen (5 mg/mL); and store it at -20 °C or -80 °C overnight. Centrifuge the samples at 20,000 × g for 20 min at 4 °C, wash the RNA pellet twice with 80% ethanol, air-dry the RNA for 5 min, and resuspend it in an appropriate volume of nuclease-free water. Determine the RNA concentration using a NanoDrop.

Adenylation of the 3' HD Adapter

1. The adenylation of the 3' HD adapter should be performed according to the manufacturer's instructions for the 5' DNA Adenylation Kit.
2. Incubate the reaction at 65 °C for 1 h.
3. Incubate the reaction mix at 85 °C for 5 min to inactivate the Mth RNA ligase, and then chill on ice.
4. Clean up the adenylated adapter using the Zymo Oligo Clean and Concentrator using the manufacturer's instructions.
5. Determine the concentration of the adenylated adapter by Nanodrop and adjust it to a final concentration of 10 μM. The molecular weight for the 3' HD adapter is 8015 g/mol.
6. Run the adenylated and non-adenylated adapter on the same 15% urea polyacrylamide gel. To prepare a 15 mL urea polyacrylamide gel, first prepare a 5 mL solution of 3.5 Ml nuclease-free water and 1.5 mL of 5× TBE in a 50-mL falcon
tube. Weigh out 6.3 g of urea and dissolve in the water/TBE solution at 37–42 °C. Warming the mixture in the microwave is not advised as the Falcon tube can explode. Add 5.5 mL of 40% acrylamide/bis solution (19:1), followed by 7.5 μL of N,N,N',N'-tetramethylethylenediamine (TEMED) and 150 μL of 10% ammonium persulfate (APS). Adjust the final volume to 15 mL with nuclease-free water. The adenylated adapter can be stored at -80 °C.

3' HD Adapter Ligation

1. Thaw 50% PEG 8000 in a 37 °C heat block and then keep at room temperature for the remainder of the protocol.
2. Set up heat blocks at 26 °C and 70 °C.
3. Mix 11.25 μL of the RNA sample with 1 μL of 10 μM pre-adenylated 3' HD adapter.
4. Denature the RNA and adapter mixture at 70 °C for 2 min and then chill on ice.
5. Add the following ligation reaction mix to the denature RNA and adapter mixture.
6. Incubate the reaction mix at 26 °C for 2 h.
7. Clean up the 3' end ligation reaction using the Zymo RNA Clean and Concentrator following the protocol provided by the manufacturer. In the final stage of the kit clean-up, elute the RNA in 12.1 μL of nuclease-free water and keep it on ice.

Removal of Excess 3' HD Adapter

1. Deadenylate the remaining 3' HD adapter using the deadenylase from NEB.
2. Incubate the above reaction mix at 30 °C for 30 min.
3. Stop the reaction by adding 4 μL of 25 mM EDTA and place the tube on ice immediately.
4. Add 2 μL of 0.5 M Tris–HCl (pH 9.0), 7 μL of 50 mM MgCl2, and 1 μL of RecJ Exonuclease to the tube. Mix well.
5. Incubate the exonuclease reaction at 37 °C for 30 min.

5' HD Adapter Ligation

1. Denature 1 μL of 20 μM 5' HD adapter at 70 °C for 2 min and chill it on ice.
2. Add the denatured 5' HD adapter to the exonuclease reaction mix.
3. Add the reagents into the tube sequentially, and mix by pipetting.
4. Incubate the 5' ligation reaction at 26 °C for 2 h.
5. Clean up the 5' ligation reaction using the RNA Clean and Concentrator Kit following the manufacturer's instructions; in the last step, elute the ligation mixture in 30 μL of nuclease-free water and place the reaction on ice.

cDNA Synthesis and Amplification

1. Set up the following cDNA synthesis reaction and mix thoroughly. Incubate at 37 °C for 20 min.
2. Inactive the reaction by incubating for 15 min at 85 °C and then chill on ice. The cDNA generated from this step can be stored at -20 °C or -80 °C indefinitely.
3. Set up the following PCR reaction mixture to amplify the cDNA library.
4. Run the PCR reaction at the following conditions. The final products can be stored at -20 °C indefinitely.

Gel Electrophoresis and Extraction of PCR Products

1. Run the PCR products on a native 8% polyacrylamide gel and load a 20-bp DNA ladder for size determination. We used the Mini-PROTEAN III system for the electrophoresis.
2. Run the gel at 120 V for 1.5–2 h in 0.5× TBE.
3. Stain the gel with SYBR® Gold and visualize on a scanner or UV box.
4. Cut out the DNA bands around 145–150 bp with a sterile razor—this contains the cDNA library of sRNAs.
5. Place the gel into a "gel breaker tube" and centrifuge at 20,000 × g for 5 min. We make these tubes by puncturing the base of a 0.5-mL Eppendorf tube with a BD Microlance 3 0.8 mm × 16 mm needle.
6. Elute the cDNA library in 1× NEB restriction enzyme buffer 2 by shaking the gel slurry overnight at 4 °C.
7. Add the gel slurry into a Spin-X nonsterile 45-μm column and centrifuge at 650 × g for 5 min at room temperature to remove gel debris. It helps to cut the tip of the pipette first to widen the aperture before loading into the column.
8. Concentrate the eluted cDNA library by ethanol precipitation overnight at -80 °C using the following ratios: For 300 μL of eluate, add 2 μL of GlycoBlue (5 mg/mL), 30 μL 3 NaOAc, and 975 μL of absolute ethanol. Leave the mixture at -20 °C or -80 °C overnight.
9. Centrifuge at 20,000 × g for 20 min at 4 °C.
10. Remove the supernatant and wash the pellet with 80% ethanol.
11. Air-dry for 5–10 min and resuspend in 12 μL of nuclease-free water.
12. Run the product on a second 8% polyacrylamide gel and repeating steps 1–11 of this section to remove any other products of undesired size.
13. Send the cDNA library for sequencing. For sRNA libraries we typically sequence at a standard depth of 20 million reads per sample.

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