Metatranscriptomics sample preparation protocol w/ScriptSeq
Thaw 2-ml screw-cap tube on ice (should take a while).
If using sterivex filters, follow notes in annotations.
Lyse the cells on the filters: Remove the 300 µl RNAlater carefully and discard.
Add 750 µl (1.5mL)* Lysis/Binding buffer, vortex vigorously to completely lyse the cells.
Add 75 µl (150 µl)* miRNA Homogenate Additive.
Vortex to mix.
Leave the mixture on ice for 10 min.
Transfer the 825 µl lysate to a new Non-stick RNase-free tube or a 2 mL tube with o-ring (might need to spin the tubes to get as much lysate out as possible).
Add 750 µl Acid-Phenol:Chloroform, vortex for 30-60 sec to mix.
Centrifuge for 5 min at maximum 10,000 X g at RT.
Carefully remove the top aqueous phase w/o disturbing the lower phase, transfer it to a fresh tube.
Pre-heat Elution Solution (or Nuclease-free water) to 95ºC.
Add 1.25 volumes of RT 100% ethanol to the aqueous phase, mix well, load to a Filter Cartridge in the collection tube (provided).
Centrifuge for 15 sec at 10,000 X g, discard the flow-through.
Apply 700 µl miRNA Wash Solution 1 to the Filter Cartridge and centrifuge for 10 sec at 10,000 X g, discard the flow-through.
Apply 500 µl Wash Solution 2/3, spin 15 sec at 10,000 X g, and discard the flow-through.
Repeat step 14.
Put the Filter Cartridge back in the tube, centrifuge for 1.5 min at 10,000 X g to remove residual fluid from the filter.
Transfer the Filter Cartridge into a fresh collection tube, apply 50 µl pre-heated (95 ºC) Elution Solution (0.1 mM EDTA) to the center of the filter.
Wait ~1 minute.
Spin for 30 sec at 10,000 X g. Repeat steps 17-19 with another 50 µl pre-heated (95ºC) Elution Solution.
Add 1/10th volume of DNase I buffer (~10 µl) and 2 µl DNase I (2U/µl), gently mix and incubate at 37 ºC for 30 min.
If RNeasy MinElute is to be used immediately after this, there is no need to use the DNase inactivation reagent – go directly to the RNeasy MinElute Procedure.
Add 0.1 volume (10 µl) of DNase inactivation reagent, mix well, incubate at RT for 2 min, flicking the tube occasionally.
Centrifuge for 1.5 min at 10,000 X g, transfer RNA to a fresh new 0.5 ml non-stick Rnase-free tube, store in –70 ºC.
Pre-heat elution buffer or water to 40°C.
Combine two 100 µl DNase-treated RNA samples in a 2ml Nonsticky RNase-free tube (~200 µl total).
Add 700 µl RLT buffer, and mix with by pipeting.
Add 500 µl 100% ethanol, mix thoroughly by pipetting.
DO NOT CENTRIFUGE.
Immediately load 700 µl onto an RNeasy MinElute Column in a 2 ml tube, close the tube gently, centrifuge for 15 s at ≥ 8,000 X g. Discard flow through.
Repeat step 29 to process the remaining sample volume through the column.
Transfer the spin column to a new 2 ml collection tube.
Pipet 500 µl buffer RPE onto the column, close tube gently.
Centrifuge for 15 s at ≥ 8,000 X g to wash.
Discard the flow-through.
Reuse the collection tube.
Add 500 µl of 80% ethanol to the column, close tube gently.
Centrifuge for 2 min at ≥ 8,000 X g, discard flow-through.
Transfer the column to a new 2 ml collection tube.
OPEN the cap of the tube, and centrifuge at 12,000 X g for 5 min, discard flow-through.
Elute with 25 µl pre-heated nuclease-free water or RNA storage buffer.
Transfer the spin column to a new 1.5 ml tube, pipet water or buffer directly onto the center of the silica-gel membrane, wait for 2 min at RT, close gently.
Centrifuge for 1 min at 12,000 X g to elute.
Repeat elution using 15 µl of water or buffer.
Store elutions at -80°C or continue with quantification.
Depending on your application, the recommended option for RNA quantification is:Bioanalyzer (Agilent) using the RNA 6000 Pico total RNA kit: quantitative range = 0.05-5.0 ng / µl (dilute samples 1:10 and 1:100 prior to analysis), allows visualization of RNA size distribution, less accurate, ~1 hr processing time.
Order primers (found in guidelines).
PCR amplify rRNA.Template DNA100 ngBact 23S (35 cycles)All other primers (35 cycles)Herculase 5X buffer10µl95°C    2 min95°C    2 mindNTP (10mM)1.25µl95°C    20 sec95°C    20 secF primer (10µM)1.25µl39°C    20 sec55°C    20 secR primer (10µM)1.25µl72°C    2 min72°C    2 minHerculase pol1µl72°C    3 min72°C    3 minH2OTo 50µl.
Purify PCR products using the QIAquick PCR purification kit (Qiagen)** with elution in 50 ul elution buffer and quantify DNA concentration using the Nanodrop (Nanodrop is fine here, as the PCR products should be at high conc.
(>100 ng/µl)).
Prepare separate reactions for all probes.
For a standard 20 µl reaction*:PCR amplicons (step B1, suggest 250-500 ng)*1µlATP (75 mM)2 µlGTP (75 mM)2 µlCTP (75 mM)1.5 µlUTP (75 mM)1.5 µlBiotin-11-CTP (10 mM, Roche 04739205001)3.75 µlBiotin-16-UTP (10 mM, Roche 11388908910)3.75 µl10X buffer2 µlSUPERase•InTM RNase inhibitor0.5 µlT7 RNA polymerase2 µl.
At room temperature (not on ice, as spermidine in the reaction buffer may cause DNA precipitation), mix reagents in the order listed.
Incubate at 37ºC for 4-6 hrs.
After incubation, add 1 µl DNase I (included in the MEGAscript kit) to remove the DNA template.
Incubate at 37ºC for an additional 30 min.
Purify synthesized RNA using the MEGAclearTM kit, with elution in 50 µl elution solution.
Quantify RNA concentration using either RiboGreen or Nanodrop (Nanodrop should work fine as RNA samples should be at high concentration).
Store probes at -80°C.
Bead washing (do this before or during the hybridization step).
For each sample, transfer 400 µl of beads* to a 1.5 ml tube.
Wash 1: bind beads to magnetic separation rack (takes ~2 min), pipet off and discard supernatant, re-suspend beads in equal volume of 0.1N NaOH (deactivates bead-associated RNases), and quick vortex to re-suspend, spin down, re-bind to magnet and pipet off supernatant.
Do this step quickly.
Wash #2: Repeat wash in step57 using 1X SSC buffer.
On the 3rd wash, leave beads in buffer on ice until hybridization is complete.
Wash #3: Repeat wash in step57 using 1X SSC buffer and leave beads in buffer on ice until hybridization is complete.
Hybridization.
For a 50 µl reaction*, prepare in a PCR tube: Template, total RNA (ideally 250-500 ng in <36.5 ul)X µlaRNA 16S probe (500-1000 ng)**X µlaRNA 23S probe (500-1000 ng)**X µlSUPERase•In RNase inhibitor1 µl20X Sodium chloride-citrate (SSC) buffer (RNase free)2.5 µlFormamide (100%)***10 µlNuclease-free water (if necessary)To 50 µl.
In a thermal cycler, incubate under the following conditions:  5 min at 70ºC Rampdown to 25°C using 5°C increments for 1 min each.
Remove the reaction and let sit at RT for 2-5 min.
Bead binding.
While the hybridization reaction is at RT, capture the pre-aliquoted beads (400 µl per sample) on the magnetic rack, pipet off the supernatant, and remove the beads from the rack.
Dilute the hybridization reaction in PCR tubes to 100 µl using 20% formamide in 1X SSC.
Add the hybridization reaction (now 100 µl) to the dried beads.
Incubate at RT for 10 min, with occasional flicking to mix.
Bead removal.
Quick spin the tubes.
Capture the beads on the magnetic rack (2-3 min).
Transfer the non-rRNA-containing supernatant to a 1.5 ml tube using a P200 pipettor.
Re-suspend remaining beads with 100 µl 1X SSC, capture beads as above, transfer supernatant to the same 1.5 ml tube (200 µl total volume).
Purify subtracted RNA (200 µl) to remove formamide.
Use the RNeasy MinElute kit.
Elute in 15 µl and 5 µl pre-heated to 50 °C (combine the elutions).
Run 1 µl of purified RNA (diluted 1:10 and 1:100) on the Bioanalyzer to confirm rRNA subtraction and probe removal.
Store RNA at -80°C for downstream applications.
If needed, speedvac the subtracted RNA to maximum volume of 9 µl.
Use all of the template material remaining after the rRNA-subtraction step.
In a 0.2 mL PCR tube assemble the following reaction:  Nuclease-free waterx µlrRNA-subtracted RNAy µlRNA fragmentation solution1.0 µlcDNA synthesis primer2.0 µlTotal reaction volume12 µl.
Fragment RNA by incubation at 85 ºC for 5 minutes in thermocycler with heated lid.
Stop fragmentation reaction by placing the tube on ice.
On ice preare the cDNA synthesis master mix, volumes below are per rxn: cDNA synthesis premix, 3 µl; 100 mM, DTT, 0.5 µl; StarScript Reverse Transcriptase, 0.5 µl; Total volume, 4 µl.
Gently but thoroughly mix the cDNA synthesis master mix by pipetting.
Add 4 µl of the cDNA synthesis master mix to each reaction on ice from Part C1 and mix by pipetting.
Incubate at 25 °C for 5 minutes followed by 42 °C for 20 minutes.
Cool the reactions to 37 °C and pause the thermocycler.
Remove one reaction at a time from the thermocycler, add 1.0 µl of Finishing solution and mix gently by pipetting.
Return each reaction to the thermocycler before proceeding to the next.
Incubate at 37 °C for 10 minutes.
Incubate each reaction at 95°C for 3 minutes, then cool reactions to 25 °C and pause the thermocycler.
During the 95 °C incubation prepare the terminal tagging master mix described in C3.
On ice, prepare the terminal tagging premix by combining the following on ice: Terminal tagging premix, 7.5 µl; DNA polymerase, 0.5 µl; Total volume 8 µl.
Remove one reaction at a time from the thermocycler (25 °C paused) and add 8 µl of the terminal tagging master mix.
Gently mix by pipetting.
Return each reaction to the thermocycler before proceeding to the next.
Incubate each reaction at 25 °C for 15 minutes.
Incubate each reaction at 95 °C for 3 minutes.
Then cool to 4 °C on ice or in thermocycler.
Use the Agencourt AmpureXP system to purify the cDNA with 1.8X purification.
Warm Ampure beads to room temperature for 30 minutes.
Prepare 400 µl fresh 80% ethanol at room temperature for each sample.
Add 45 µl of beads to each microfuge tube containing the di-tagged cDNA from part 3C.
Mix thoroughly by pipetting 10 times.
Transfer volume to 1.5 mL tube.
Incubate at room temp for 15 minutes.
Place tubes in magnetic stand at room temp for at least 5 minutes until liquid clear.
Remove and discard supernatant using a pipet, some liquid may remain in tube.
Ethanol Wash #1: With tubes on stand, add 200 µl 80% ethanol to each tube without disturbing the beads.
Ethanol Wash #1: Incubate for 30 seconds, then remove and discard supernatant.
Ethanol Wash #2: With tubes on stand, add 200 µl 80% ethanol to each tube without disturbing the beads.
Ethanol Wash #2: Incubate for 30 seconds, then remove and discard supernatant.
Allow tubes to air-dry on magnetic stand for 15 minutes at room temp.
Add 24.5 µl of nuclease-free water to each tube and remove from magnetic stand.
Thoroughly resuspend beads by pipetting 10 times.
Incubate tube at room temperature for 2 minutes.
Place the tube on magnetic stand at room temp for at least 5 minutes, until liquid clears.
Transfer 22.5 µl supernatant, which contains di-tagged cDNA, from each tube to a new 0.2 mL PCR tube, place on ice.
In a 0.2 mL PCR tube containing 22.5 µl of di-tagged cDNA from C4, add on ice: FailSafe PCR Premix E, 25 µl; Forward PCR primer, 1 µl; Reverse or ScriptSeq index primer, 1 µl; FailSafe PCR Enzyme (1.25 U), 0.5 µl; Total Volume, 50 µl.
Perform PCR: Denature ds DNA at 95 °C for 1 minute.
Followed by 12-15 cycles of: 95 °C for 30 seconds; 55 °C for 30 seconds; 68 °C for 3 minutes.
Finish with 68 °C for 7 minutes.
Bring thermocycler temperature down to 4 °C.
After PCR complete, proceed immediately to purification step C6.
Use the Ampure XP system to purify the PCR reaction to remove the primer-dimers that can occur during PCR.
Warm Ampure beads to room temperature for 30 minutes.
Prepare 400 µl fresh 80% ethanol at room temperature for each sample.
Add 50 µl of beads to each tube containing amplified library from part C5.
Mix thoroughly by pipetting 10 times.
Transfer each 100 µl volume to 1.5 mL tube.
Incubate at room temp for 15 minutes.
Place tubes in magnetic stand at room temp for at least 5 minutes until liquid clear.
Remove and discard supernatant using a pipet, some liquid may remain in tube.
Ethanol Wash #1: With tubes on stand, add 200 µl 80% ethanol to each tube without disturbing the beads.
Ethanol Wash #1: Incubate for 30 seconds, then remove and discard supernatant.
Ethanol Wash #2: With tubes on stand, add 200 µl 80% ethanol to each tube without disturbing the beads.
Ethanol Wash #2: Incubate for 30 seconds, then remove and discard supernatant.
Allow tubes to air-dry on magnetic stand for 15 minutes at room temp.
Add 20 µl of nuclease-free water to each tube and remove from magnetic stand.
Thoroughly resuspend beads by pipetting 10 times.
Incubate tube at room temperature for 2 minutes.
Place the tube on magnetic stand at room temp for at least 5 minutes, until liquid clears.
Transfer the supernatant, which contains the RNA-Seq library, from each tube to a new tube.
Make 1:10 and 1:100 dilutions for Bioanalyzer analysis of average library size.
Perform Picogreen to get accurate quantification of library concentration.
At this point, the samples are ready for pooling (check for compatible indices) and sequencing according to the latest MiSeq/NextSeq sequencing protocol.
For v2 sequencing kits with the MiSeq and NextSeq, we recommend a final pooled library concentration of 6 pM and 1.0 pM respectively.
