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 .
