Metatranscriptomic Sequencing Protocol

RNA Extraction

RNA Extraction from Soil or Sediment

1. 0.5 g (wet weight) of soil is added to a 2 ml microcentrifuge tube.
2. 0.5 ml of hexadecyltrimethylammonium bromide (CTAB) buffer and 0.5 ml of phenol–chloroform–isoamyl alcohol (25:24:1) (pH 8.0) are added to each extraction.
3. Shake tubes in a bead beater system for 30 s at 5.5 m/s.
4. The aqueous phase is separated by centrifugation (16,000 × g) for 5 min at 4°C.
5. The aqueous phase was added to an equal volume of chloroform-isoamyl alcohol (24:1).
6. Sample is centrifuged at (16,000 × g) for 5 min at 4°C.
7. DNA and RNA are precipitated from the aqueous layer with 2 volumes of 30% (wt/vol) polyethylene glycol 6000–1.6 M NaCl for 2 h at room temperature, followed by centrifugation at 18000 × g, 4°C for 10 min.
8. The nucleic acid pellet is then washed with ice-cold 70% (vol/vol) ethanol by centrifugation at 10,000 × g for 20 min.
9. The nucleic acid is then air-dried for 15 min prior to resuspension in 1000 ml of RNase-free Tris–EDTA buffer.
10. 100 ml of the total RNA should be purified with b-mercaptoethanol added to the RLT buffer.
11. Approximate RNA concentration is determined by spectrophotometry and checked for rRNA integrity. The integrity of rRNA was demonstrated by highly defined, discrete rRNA peaks, with the 23S rRNA peak being 1.5–2 times higher than the 16S rRNA peak.
12. DNA contamination was removed from total RNA samples by treating with the Turbo DNA-free enzyme.

RNA Extraction from Seawater

1. Filter 10–15 L of seawater through a 140 mm diameter, 1.6 mm GF/A filter, to reduce eukaryotic cell abundance and maximize the proportion of prokaryotic cells.
2. Apply filtrate directly to a 0.22 mm Sterivex filter.
3. Following filtration, each Sterivex was pumped dry and frozen in liquid nitrogen.
4. After thawing on ice, add 1.6 ml of SET lysis buffer directly on top of Sterivex using a 2.5 ml syringe.
5. Add 180 ml of fresh lysozyme and seal the Sterivex.
6. Incubate at 37°C for 30 min.
7. Add 200 ml of SDS.
8. Add 55 ml of 20 mg/ml fresh proteinase K.
9. Incubate at 55°C for 2 h.
10. Withdraw lysate into a 5-ml syringe.
11. Add 1 ml of fresh SET buffer to Sterivex and rotate to rinse.
12. Withdraw rinse buffer into the same 5 ml syringe.
13. Add lysate to 15 ml tube containing 2 ml of phenol–chloroform–isoamyl alcohol (25:24:1), pH 8. Shake gently until mixed and then centrifuged at 1,500 × g for 5 min.
14. Add an additional 2 ml of phenol–chloroform–isoamyl alcohol (25:24:1). Shake gently until mixed and centrifuge at 1500 × g for 5 min.
15. Add 2 ml of chloroform–isoamyl alcohol (24:1). Shake gently until mixed and centrifuge at 1500 × g for 5 min.
16. Decant aqueous phase to a sterile and DEPC-treated (if RNA needed) 20-ml centrifuge tube and add 0.5 V of 7.5 M ammonium acetate. Mix briefly and then add 2.5 V of pure ethanol.
17. Mix and leave at −20°C for >1 h (overnight is fine).
18. Centrifuge at 10,000 × g for 30 min at 4°C and decant ethanol.
19. Add 2 ml of 80% ethanol and rinse tube, then centrifuge at 10,000 × g for 20 min at 4°C and decant ethanol, and repeat.
20. Decant ethanol and leave inverted for 15 min in fume hood.
21. Suspend the pellet in 200 ml of DEPC-treated sterile water. Leave on ice for approximately 1 h with frequent finger-tapping to rinse tube walls.

mRNA Enrichment Techniques

1. Total RNA was applied to the subtractive hybridization method to remove rRNA from the mRNA. The manufacturer's instructions provide sufficient detail to carry out this procedure.
2. mRNA was eluted in 25 ml of TE buffer
3. Remove small RNAs and small contaminants, as the manufacturer's instructions. Purified mRNA was eluted in 10 ml of DEPC-treated water.
4. mRNA was then reverse-transcribed to cDNA using enzyme with random primers following the manufacturer's instructions for random primer transcription.
5. The cDNA was treated with RNase to remove trace RNA contaminants, with incubated at 37°C for 20 min.
6. 1 ml of cDNA was then randomly amplified. Ideally, this reaction is performed 10×, and then these replicates are pooled to remove potential random amplification bias inherent in multiple displacement amplification technology.
7. Amplified samples are treated with S1 nuclease at 2 m/mg cDNA. The reaction is incubated in supplied buffer at 37°C for 30 min. The reaction is stopped by adding 20 mM final concentration EDTA and then cleaned up.
8. cDNA was nebulized and then cleaned with AMPure beads.
9. cDNA was then sequenced.

Reference:

1. Gilbert J A, Laverock B, Temperton B, et al. Metagenomics[M]//High-Throughput Next Generation Sequencing. Humana Press, Totowa, NJ, 2011: 173-183.