Extraction of DNA and RNA for Metagenome and Metatranscriptome Sequencing Protocol

Simultaneous DNA and RNA Extraction from Samples

Co-Extraction of DNA and RNA

1. For each extraction, 3 × 50-mL Greiner and 4 × 15-mL Greiner tubes are needed and should be labeled carefully.
2. Place 2 g of each size (2 and 3 mm) fresh and sterile glass beads into each fresh 50-mL Greiner tube.
3. To prepare the extraction mixture for one extraction, mix 7.5 mL extraction buffer with 0.2 mL 20% SLS solution. Always calculate an overhead of one additional sample for master mixes.
4. Prepare buffer saturated phenol and add 10 mg of 8-hydroxyquinoline per 10 mL Roti®-Aqua-Phenol.
5. Use sterile scissor and forceps to cut the frozen filter sandwich into short pieces. Place the filter pieces in the Greiner tube.
6. Add 5 mL of the extraction mixture.
7. Add 5 mL of buffer-saturated phenol.
8. Vibrate the mixture with 4 m/s for 60. Centrifuge for 20 min at 2000× g at 4 °C. During centrifugation, the mixture separates into a lower phenol phase with glass beads, an interphase, and an upper aqueous phase.
9. Transfer the upper aqueous phase containing the RNA to a fresh 15-mL Greiner tube and store on ice.
10. Add 2 mL of the extraction mixture and 2 mL of buffersaturated phenol to the phenolic phase for repeated phenol extraction.
11. Mix thoroughly by vortexing the capped tube and centrifuge again for 20 min at 2000× g at 4 °C.
12. Transfer the upper aqueous phase to the already collected aqueous phase from step 8. The volume of the pooled aqueous phases is about 5 mL. Store on ice.
13. For DNA isolation, add 5 mL Tris-base to the phenolic phase and mix well. Store at 4 °C for at least 40 min but not longer than 3 h.
14. Continue with RNA isolation.

RNA Isolation

1. Add 0.1 volumes of 3 M sodium acetate to the Greiner tube containing the pooled aqueous phases from the extraction and phase separation procedure.
2. Add 5 mL of chloroform-isoamyl alcohol.
3. Mix vigorously by vortexing and centrifuge for 10 min at 10,020× g and 4 °C to separate the phases.
4. Thaw glycogen (35 μg/mL) on ice.
5. Transfer the aqueous phase to a fresh Greiner tube and repeat steps 2 and 3.
6. Transfer the aqueous phase to a fresh Greiner tube and add a 1/700 volume of glycogen.
7. Mix vigorously and add one volume of isopropanol to precipitate the RNA.
8. Mix vigorously and incubate samples at -20 °C overnight.
9. Continue with DNA isolation.

DNA Isolation

1. Continue with DNA isolation by vigorously mixing the Greiner tube containing the extracted DNA and centrifuge for 15 min at 2000 × g and 4 °C.
2. Transfer the upper aqueous phase to a fresh Greiner tube.
3. Add 2 mL 1 M Tris-base to the lower phenolic phase and repeat mixing and centrifugation for 15 min at 2000× g and 4 °C.
4. Transfer the upper aqueous phase to the aqueous phase already collected in step 2. Add 5 mL of chloroform-isoamyl alcohol to the tube.
5. Mix by vortexing and centrifuge for 20 min at 10,000 × g and 4 °C to separate the phases.
6. Transfer the upper phase to a fresh Greiner tube and repeat steps 4 and 5.
7. Add 0.1 volume of 3 M sodium acetate and 1/700 volume of glycogen.
8. Mix vigorously. Add 2.5 volumes of ice-cold pure ethanol to precipitate the DNA.
9. Mix vigorously and incubate samples at -20 °C overnight.

Washing and Resuspension of RNA and DNA

1. Pellet the precipitated nucleic acids by centrifugation at maximum g for 30 min and 4 °C.
2. Wash the pellets twice with 1 mL of ice-cold 80% ethanol. Centrifuge at maximum g for 15 min and 4 °C.
3. Dry pellet for about 10 min at room temperature and dissolve the DNA or RNA in 200 μL 1× TE buffer.

DNA and RNA Purification

Removal of Residual RNA from DNA Samples

1. Add 1 μL RNase A to the extracted DNA.
2. Incubate at 37 °C for 1 h.
3. Purify the DNA using the Gene Read Size Selection Kit.
4. Elute DNA with 2× 25 μL pre-warmed DEPC-treated water.

RNA Purification

1. Add 700 μL RLT buffer and 7 μL ß-ME. Mix well.
2. Add 500 μL of 96–100% ethanol to the diluted RNA. Mix well. Do not centrifuge. Proceed immediately.
3. Transfer 700 μL sample to an RNeasy Mini spin column placed in a 2-mL collection tube. Close the lid gently and centrifuge for 15 s at 10,020 ×g. Discard the flow-through.
4. Repeat step 3 once.
5. Place the RNeasy Mini spin column in a new 2-mL collection tube.
6. Add 500 μL Buffer RPE to the spin column.
7. Close the lid gently and centrifuge for 15 s at >10,020× g. Discard the flow-through.
8. Add 500 μL of 80% ethanol to the RNeasy Mini spin column. Close the lid gently and centrifuge for 2 min at 10,020× g.
9. Place the RNeasy Mini spin column in a new 2-mL collection tube. Open the lid of the spin column and centrifuge at full speed for 5 min. Discard the flow-through and collection tube.
10. Place the RNeasy Mini spin column in a new 1.5-mL collection tube. Elute RNA two times with 50 μL DEPCtreated H₂O (~95 μL eluate) for 1 min at full speed at 4 °C.
11. Mix 5 μL of the purified RNA with 5 μL 2× RNA Loading Dye and control the success of the RNA extraction and purification by running on a 2% agarose gel.

DNA Digestion

1. If nucleic acid solution concentration is larger than 200 ng/μL, dilute with DEPC-treated water.
2. Add 1/10 volume of 10× TURBO DNase Buffer (supplied) to RNA sample.
3. Add 1/40 volume of RiboLock RNase Inhibitor (final concentration 1 U/μL).
4. Add 1 μL TURBO DNase (2 U) per 10 μg of RNA.
5. Incubate at 37 °C for 30 min.
6. Add 0.1 volumes of DNase Inactivation Reagent and mix well.
7. Incubate at room temperature for 5 min. Mix occasionally.
8. Centrifuge at 10,000 × g for 1.5 min and transfer the RNA to a fresh tube.
9. Perform 16S rRNA control PCR. Repeat steps 3–8 if necessary.
10. Purify the RNA with the RNeasy MinElute kit as recommended.
11. Elute the RNA in 24 μL DEPC-treated water.

Control PCR for Residual DNA

1. Combine the following ingredients in a sterile DNA-free 0.2-mL PCR tube: 0.25 μL 5× Phusion HF buffer, 0.5 μL dNTP mix (10 mM each), 0.75 μL 25 mM MgCl₂, and 0.25 μL HighFidelity Phusion DNA polymerase (2 U/μL). Add up to a final volume of 24 μL with sterile RNase-free H₂O.
2. Add 1 μL of DNase-treated RNA, to the mixture.
3. Perform negative controls using the reaction mixture without template and positive controls using E. coli DH5alpha DNA.
4. Use the following thermal cycling scheme: Initial denaturation at 98 °C for 30 s, 25 cycles of denaturation at 98 °C for 10 s, annealing at 62 °C for 30 s, followed by extension at 72 °C for 30 s. Final extension at 72 °C for 5 min.
5. Control the success of the DNA digestion by running 5 μL of the PCR reaction on a 2% agarose gel.

First- and Second-Strand Synthesis

1. Mix 8 μL of pure RNA with 1 μL of reverse primer (20 μM) without MiSeq adaptor of the desired amplicon, 1 μL dNTPs (10 mM each), and 3 μL sterile RNase-free water.
2. Incubate at 65 °C for 5 min.
3. Quick-chill on ice for at least 1 min.
4. Add in the following order: 4 μL 5× reaction buffer, 1 μL 0.1 M DTT, 1 μL RiboLock Inhibitor and 1 μL SuperScript™ III RT (200E).
5. Vortex gently and collect the reaction by brief centrifugation.
6. Incubate at 55 °C for 1 h.
7. Incubate at 70 °C for 15 min.
8. Add 0.5 μL RNase H (5 U/μL).
9. Incubate for 15 min at 37 °C.
10. Incubate for 10 min at 65 °C.
11. Proceed with desired amplicon PCR by using 1 μL of generated cDNA or store samples at -20 °C until further usage.