TITLE: Total RNA Protocol (extraction, quantification and Illumina library preparation)
AUTHORS: Antonio Mondini, Morten Dencker Schcostag, Lea Ellegard-Jensen, Toke Bang-Andreasen, Muhammad Zohaib Anwar, Cristina Purcarea, Carsten Suhr Jacobsen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">This experimental procedure was designed to isolate total the RNA aiming to obtain intact and high-quality integrity (RIN>7) for further next generation sequencing analysis. The protocol was developed to analyse the environmental microbial active community providing a clear overview of the microbial transcriptional activity in a specific time or after a different condition exposition sets, such as the investigation of thermal adaptation after heat-shock. The procedure is specifically designed to extract total-RNA from water samples although it can be used for various environmental samples as done in  Schostag et al. and Bang-Andreasen et al. 2019 in soil with  similar accuracy results. The total-RNA yield was >1000 ng/mL with a RIN ranging from 7 to 8.5. The final Illumina library preparation yield ranged between 15 and 31 ng/µL.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>This protocol is a compiled by using the Qiagen RNeasy® PowerSoil® Total RNA Kit with additional steps according the DNeasy Max®Kit (Qiagen), NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, Inc), KAPA Library Quantification Kit for Illumina® Platforms (Kapa Biosystems), Qubit 2.0 (Thermo Fisher Scientific, Hvidovre, Denmark), Bioanalyzer® (Agilent Technologies, Inc.) sample Purification Beads (New England Biolabs) and Liquid G2 DNA/RNA Enhancer (Ampliqon) manufacturer protocols.</span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span>  </span></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/179d3a3f5f84aee72da371b09e34edcbb804e894a929d767250332df1b71a3cd/bmrd7ptn.png" /></div></div>

[STEPS]
?. [Water samples filtration]
Once completed the filtration, transfer the filters in the 15 mL tubes with beads (provided in the RNeasy®kit).
Transfer the filters carefully folding it to fit in the 15mL tubes using two forceps and letting the surface with visible filtered particles inward the tube. Push carefully down to the beads.
No more than 2 filters can be added to a single tube
?. [RNA Isolation]
Add G2 and snap freeze immersing the bead tubes 15 seconds in liquid nitrogen keeping the lid slightly open
Add 500µLof G2 each filter. If a thick sediment layer accumulates on the filter, it has to be weighed and 500 µL of G2 added each 0.25 grams of sediments. No more than 1 gram can be added to the tube. Addition of more soil/sediments can reduce the lyse cells efficiency. For more info on G2 test see appendix 1   . Alternatively, to liquid nitrogen, tubes can be freeze O/N at -20°C
?. [RNA Isolation]
Immediately add 2.5 ml of PowerBead Solution
?. [RNA Isolation]
Add 0.25 ml of Solution SR1
?. [RNA Isolation]
Add 0.8 ml of Solution IRS
?. [RNA Isolation]
Add 3.5 ml of phenol/chloroform/isoamyl alcohol (pH 6.5) and vortex until the solution is homogeneous.
After vortexing the solution should have a muddy consistency. At this stage the tubes have to be placed outside the Microbiological hood and to avoid phenol chloroform diffusion in the air is preferably to seal the tubes using parafilm
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?. [RNA Isolation]
Place the PowerBead tube horizontally on a Vortex Adapter (cat. no. 13000-V1-15) and vortex at maximum speed for 20 minutes at
0 Room temperature
This vortexing time was specifically adapted for tubes containing filters with the addition of Phenil chloroform and previously snap freezed in Liquid Nitrogen. For different samples vortex for 15 minutes according to the manufacturer protocol
?. [RNA Isolation]
Remove PowerBead tube from the shaker and centrifuge  for 10 minutes at 2,500 x g
0 Room temperature
For different centrifuge unit see appendix 2
?. [RNA Isolation]
Transfer the upper aqueous phase (avoid the interphase and lower phenol layer) to a clean 15 ml Collection Tube (provided) using a 1000µL pipette. Ensure to take only the top layer (Approx. 3 mL) leaving some aqueous phase to not disturb the interphase.
If the filters, after centrifuge, are interfering with normal procedure of pipetting or have formed agglomerates, pour the tube content (leaving behind the filters) in a new 15 mL tube and repeat the centrifugation at step 9.
?. [RNA Isolation]
Add 1.5 ml of Solution SR3 to the tube containing the aqueous phase and vortex to mix until the solution gets homogeneous
?. [RNA Isolation]
Incubate at  for 10 minutes
4 °C
?. [RNA Isolation]
Centrifuge at 2,500 x g   for 10 minutes
0 Room temperature
?. [RNA Isolation]
Transfer the supernatant, without disturbing the pellet if present (usually not present), to a new 15 ml Collection Tube (provided)
Its preferred to leave behind few microliters
?. [RNA Isolation]
Add 5 mL of Solution SR4 to the supernatant in the Collection Tube using a serological 5 mL pipette
?. [RNA Isolation]
Vortex until the biphasic layer disappear (2 cycles of 10 seconds)
?. [RNA Isolation]
Incubate at   for 30 minutes
0 Room temperature
?. [RNA Isolation]
Centrifuge at 2500 x g at  for 30 minutes
0 Room temperature
?. [RNA Isolation]
Decant the supernatant
?. [RNA Isolation]
Quickly spin down the tubes to collect remaining drops on the tubes wall
?. [RNA Isolation]
Remove the collected liquid without disturbing the pellet
?. [RNA Isolation]
Allow the tubes to dry for 5 minutes
Do not let the pellet over dry (more than 10 minutes) or will result hard to resuspend it.Pellet colors were observed in a range of light grey and more often light/dark brown.
?. [RNA Purification]
Shake Solution SR5 and add 1 ml to the 15 ml Collection Tube.
?. [RNA Purification]
Resuspend the pellet completely by repeatedly pipetting.
Usually is hard to be resuspended and using a P1000 pipette just remove the pellet from the wall and proceed to the next step.
?. [RNA Purification]
Incubate the tubes at  in a preheated thermoblock. Check on the tube every 5 min and vortex the solution. Visual inspection should reveal the total resuspension.
45 °C
?. [RNA Purification]
Repeat step 25 until the pellet is fully resuspended
?. [RNA Purification]
Once the pellet is resuspended place the JetStar Mini Column (provided capture column) in a new 15 ml tube
?. [RNA Purification]
Shake SR5 solution and add 2 mL to the RNA capture column
From this step on never allow to completely gravity flow through the column.A dry column will reduce the downstream RNA yield.Add warning here
?. [RNA Purification]
Once the SR5 solution has reached the top of the resin (1 mm thickness) in the column add the dissolved pellet to the column and allow it to gravity flow through the column
?. [RNA Purification]
When dissolved pellet has reached the top of the resin (1 mm thickness) add 1 ml of shacked SR5 solution to the capture column and allow it to gravity flow through it
?. [RNA Purification]
Once the SR5 solution has reached the top of the resin shake solution SR6 and add 1 mL to the column
?. [RNA Purification]
Immediately transfer the RNA capture column to a new clean 15 mL tube
?. [RNA Purification]
Allow solution SR6 to flow through the column. Here wait until all the column is completely dry
?. [RNA Purification]
Transfer the eluted RNA to a 2.2 mL Collection Tube (provided)
?. [RNA Purification]
Shake solution SR4 and add 1mL to the 2.2 mL collection tube. Close gently the lid and invert tube to mix 10 times.
?. [RNA Purification]
Incubate tube at   for 30 minutes
-20 °C
?. [RNA Purification]
Centrifuge the 2.2 vials at   for 15 minutes at 13,000 x g
4 °C
After centrifugation the pellet may have a light brown color or white. If the final RNA yield is low may be barely visible and in this case is recommendable to place the vials in the centrifuge memorizing the position where the centrifugation force may deposit the pellet in order to have a proper final RNA resuspension.
?. [RNA Purification]
Decant the supernatant
?. [RNA Purification]
Quickly spin down the tubes to collect remaining drops on the tubes wall
?. [RNA Purification]
Remove the supernatant using a smaller pipette (P20 or P100) without touching the pellet
?. [RNA Purification]
Air dry the pellet for 5 minutes
?. [RNA Purification]
Resuspend the RNA pellet in 40 µL of Solution SR7
?. [RNA Purification]
Split the resuspended RNA in clean RNase free 200µL PCR tubes as follows. Final Volume (FV) 40 µLTube 1 –    17 µLTube 2 –    17 µLTube 3 –   ~ 6 µL
Tube 3 will be used for RNA quantification and RIN assessment while tube number 1 will be used for the downstream DNase treatment. Tube 2 can be stored at -80°C as a back-up sample in case of problems during the downstream treatments. If a low RNA yield is occurring (according to preliminary tests) resuspend the RNA pellet (step 42) in a 20 µL final volume and split the sample as follows:   Tube 1 –  17 µL  Tube 2 –  ~3 µL
?. [RNA Purification]
Place the PCR tubes on ice and proceed to step 45 or place the samples at   for short storage and                   for longer
-20 °C
-80 °C
?. [RNA yield assessment]
Place the PCR tubes containing the eluted RNA on ice
?. [RNA yield assessment]
Determine concentration of RNA using Qubit. Take 2-4 µL from tube #3
?. [DNase treatment using DNase Max Kit]
Thaw on ice the tube #1 containing 17µL total RNA extraction from step 43
?. [DNase treatment using DNase Max Kit]
Add 2µL of 10X DNase Buffer
?. [DNase treatment using DNase Max Kit]
Add 1 µL of DNase I enzyme (10 units)
?. [DNase treatment using DNase Max Kit]
Mix by gently pipetting
Don’t vortex the DNase I enzyme or the final mixture. It will denature the enzyme
?. [DNase treatment using DNase Max Kit]
Incubate the mixture, using a thermocycler, at   for 20 minutes and proceed to the next step, the DNase removal
37 °C
?. [DNase treatment using DNase Max Kit]
Resuspend the DNase Removal resin by vortexing until the slurry is homogeneous
?. [DNase treatment using DNase Max Kit]
Add 5 µL of DNase removal resin to the mixture (step 51)
?. [DNase treatment using DNase Max Kit]
Incubate for 10 minutes at   and check on the tube every 2 minutes with resuspending the resin by gently pipetting the solution
0 Room temperature
?. [DNase treatment using DNase Max Kit]
Centrifuge the tubes at 13.000 x g for 1 minute to pellet the resin
?. [DNase treatment using DNase Max Kit]
Transfer the supernatant containing the RNA target to a new clean 200µL tube without touching or removing the resin
To avoid contaminant carryover pipette approximately 15µL
?. [DNase treatment using DNase Max Kit]
Determine the successful DNA removal procedure using Qubit or for higher detection limit perform a qPCR control. Use 1 to 5 µL from the previous step (56)
?. [DNase treatment using DNase Max Kit]
Determine concentration of RNA using Qubit. Use 1-2  µL
?. [DNase treatment using DNase Max Kit]
Proceed to the RIN assessment number or store the RNA at
-80 °C
?. [RNA quality assesment (RIN)]
Allow all reagents to equilibrate to room temperature for 30 minutes before use and protect dye and dye mixture from light.
?. [RNA quality assesment (RIN)]
Set up the priming station for Bioanalyzer® chip adjusting the base plate in C position and sliding the syringe clip up to the top position
Be sure of the clip position. Different position will lead to a negative output
?. [RNA quality assesment (RIN)]
Prepare the RNA ladder and the gel
Ladder and gel preparation described in appendix 3
?. [RNA quality assesment (RIN)]
Vortex RNA dye concentrate (blue cap vial) for 10 seconds, spin down and add 1 μL of dye into a 65 μL aliquot of filtered gel
?. [RNA quality assesment (RIN)]
Vortex solution well. Spin tube at 13000g for 10 minutes at
0 Room temperature
Use prepared gel-dye mix within one day
?. [RNA quality assesment (RIN)]
Place a new RNA chip on the chip priming station
Always insert the pipette tip to the bottom of the well when dispensing the liquid. Placing the pipette at the edge of the well may lead to poor results.
?. [RNA quality assesment (RIN)]
Pipette 9 μL of gel-dye mix in the well marked
?. [RNA quality assesment (RIN)]
Make sure that the plunger is positioned at 1 mL and then close the chip priming station
?. [RNA quality assesment (RIN)]
Press plunger until it is held by the clip
?. [RNA quality assesment (RIN)]
Wait for exactly 30 seconds then release clip
?. [RNA quality assesment (RIN)]
Wait for 5 seconds and slowly pull back plunger to 1 mL position
?. [RNA quality assesment (RIN)]
Open the chip priming station and pipette 9 μL of gel-dye mix in the wells marked with
?. [RNA quality assesment (RIN)]
Pipette 9 μL of the RNA conditioning solution (vial with white cap) into the well marked CS
?. [RNA quality assesment (RIN)]
Pipette 5 μL of RNA marker (green cap vial ) in all 11 sample wells and in the well marked
?. [RNA quality assesment (RIN)]
Pipette 1 μL of the heat denatured and aliquoted ladder in the well marked
?. [RNA quality assesment (RIN)]
Pipette 1 μL of sample in each of the 11 sample wells. Pipette 1 μL of RNA Marker  (green cap vial) in each unused sample well
It is recommended to heat denature all RNA samples before use for 2 min and 70 °C (once) and keep them on ice
?. [RNA quality assesment (RIN)]
Place the chip horizontally in the IKA vortexer and vortex for 1 min at 2400rpm.
DO NOT VORTEX FOR MORE THAN 1 MINUTE AND MORE THAN 2400rpm
?. [RNA quality assesment (RIN)]
Place the chip in the Agilent 2100 Bioanalyzer and run the instrument within 5 min
Set up the Bioanalyzer instrument prior loading the chip. Keeping loaded gel for more than 5 minutes will allow the liquid in it to evaporate leading to a poor or non-acceptable analysis.For a clear overview of the RNA integrity electropherogram output from the bioanalyzer see appendix 4
?. [Illumina library preparation]
RNA Fragmentation and PrimingThaw the RNA samples from step 58 on ice
This procedure was used for libraries without any enrichment or depletion of total RNA with RIN scores > 7 and a concentration of 100 ng purified RNA. RNA was prepared in 5 μL final volume of Nuclease-free water, to start with the first protocol step. The protocol is optimized for approximately 200 nt RNA inserts. To generate libraries with longer RNA insert sizes or lower RIN number, refer to Neb manufacturer protocol.
Assemble all the reaction on ice using a nuclease-free tubes and in a dedicated clean PCR hood. The Illumina library preparation requires 7 steps using the thermocycler with different incubation times/cycles and temperatures;it is highly recommended to create a dedicated folder in the thermocycler with saved programs as suggested in the protocol in a chronological way. See appendix 5   to set up the thermocycler
?. [Illumina library preparation]
Perform dilutions, if necessary, to obtain a 100ng of total RNA in 5µL Final Volume using new clean 200µL PCR tubes
?. [Illumina library preparation]
Set up the reaction (using the tubes from the previous step) as follows:
?. [Illumina library preparation]
Set a 20 μL pipette to 5 μL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube
?. [Illumina library preparation]
Place the sample in a thermocycler, upload and start program #1
For program #1 see appendix 5
?. [Illumina library preparation]
Once the incubation is ended, immediately transfer the tube to ice
?. [Illumina library preparation]
Keeping the tubes on ice, assemble the following reaction:
?. [Illumina library preparation]
Set a 20 μL pipette to 10 μL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube
?. [Illumina library preparation]
Place the sample in a thermocycler, upload and start program #2
For program #2 see appendix 5
?. [Illumina library preparation]
Once the incubation is ended, immediately transfer the tube to ice and proceed to next step
?. [Illumina library preparation]
Keeping the tubes on ice, assemble the following reaction:
?. [Illumina library preparation]
Set a 100 μL pipette to 40 μL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube
?. [Illumina library preparation]
Place the sample in a thermocycler, upload and start program #3
For program #3 see appendix 5
?. Once the incubation is ended proceed to the first Purification step (91.1)
?. [Purification of Double-stranded cDNA]
Resuspend the NEBNext Sample Purification Beads by vortexing until the slurry is  homogeneous
Before starting, prepare 80% ethanol; use a daily freshly prepared one. It is preferably to process no more than 6 samples at once; this will reduce the risk of over-dry the beads with a lower DNA target recovery. In this protocol the purification was performed usingNEBNext Sample Purification Beads
?. [Purification of Double-stranded cDNA]
Add 144 μL (1.8X) of resuspended beads to the tubes from step 89 (second strand synthesis, ~80 μl)
?. [Purification of Double-stranded cDNA]
Mix well on a vortex mixer
?. [Purification of Double-stranded cDNA]
Incubate for 5 minutes at
0 Room temperature
?. [Purification of Double-stranded cDNA]
Briefly spin the tube in a microcentrifuge to collect any sample on the sides of the tube
?. [Purification of Double-stranded cDNA]
Place the tube on a magnetic rack for ~5 minutes to separate beads from the supernatant
?. [Purification of Double-stranded cDNA]
Once the solution is clear, carefully remove and discard the supernatant without either removing or touching the beads
Be careful with the beads, they retain the target DNA
?. [Purification of Double-stranded cDNA]
Add 200 μL of 80% ethanol to the tube while in the magnetic rack
?. [Purification of Double-stranded cDNA]
Incubate at   for 30 seconds and then carefully remove and discard the supernatant
0 Room temperature
?. [Purification of Double-stranded cDNA]
Repeat from Step 91.8 once for a total of 2 washing steps
?. [Purification of Double-stranded cDNA]
Briefly spin the tubes and put the tubes back on the magnetic rack
?. [Purification of Double-stranded cDNA]
Remove the residual ethanol pipetting and air dry the beads for up to 5 minutes while the tube is on the magnetic rack with lid open
Do not over-dry the beads. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry
?. [Purification of Double-stranded cDNA]
Remove the tube from the magnetic rack
?. [Purification of Double-stranded cDNA]
Add 53μL of 0.1X TE Buffer (provided) to elute the DNA from the beads
?. [Purification of Double-stranded cDNA]
Mix well on a vortex mixer
?. [Purification of Double-stranded cDNA]
Quickly spin the tube and incubate for 2 minutes at
0 Room temperature
?. [Purification of Double-stranded cDNA]
Place the tube on the magnetic rack for ~5 minutes, until the solution is clear
?. [Purification of Double-stranded cDNA]
Remove 50μL of the supernatant and transfer to a clean 200µL nuclease free PCR tube without disturbing the beads pellet
If you need to stop, this is a safe stop point. Samples can be stored at -20°C
?. [Illumina library preparation]
Place the tube from step 91.18 on ice and assemble the following reaction:
Here is possible to prepare a mastermix. If a master mix is made, add 10μL of master mix to 50μL of cDNA for the End Prep reaction
?. [Illumina library preparation]
Set a 100 μL pipette to 50 μL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube
It is important to mix well. The presence of a small amount of bubbles will not interfere
?. [Illumina library preparation]
Place the sample in a thermocycler, upload and start program #4. Immediately proceed to the next step
For program #4 see appendix 5
?. [Illumina library preparation]
During the incubation, perform a 5-fold dilution of the   (red) NEBNext Adaptor in Adaptor Dilution Buffer and keep the diluted adaptors on ice
The NEBNext adaptor is provided in NEBNext oligos
?. [Illumina library preparation]
Once both the Adaptor dilution and incubation are done, assemble the ligation reaction on ice by adding the following components
Follow the order given. We do not recommend a premix
?. [Illumina library preparation]
Set a 100 μL pipette to 80 μL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube
The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance
?. [Illumina library preparation]
Place the sample in a thermocycler, upload and start program #5
For program #5 see appendix 5
?. [Illumina library preparation]
Add 3 μL    (blue cap vial) USER Enzyme to the ligation mixture after the incubation, resulting in total volume of 96.5 μL
?. [Illumina library preparation]
Set a 100 μL pipette to 80 μL and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube
?. [Illumina library preparation]
Place the sample in a thermocycler, upload and start program #6
For program #6 see appendix 5
?. Once the incubation is ended proceed to the second Purification step
?. [Purification of the Ligation Reaction]
Resuspend the NEBNext Sample Purification Beads by vortexing until the slurry is homogeneous
?. [Purification of the Ligation Reaction]
Add 87μL (0.9X) of resuspended beads to the tubes from step 100 (Ligation reaction, ~96.5μL)
?. [Purification of the Ligation Reaction]
Mix well on a vortex mixer
?. [Purification of the Ligation Reaction]
Incubate for 10 minutes at
0 Room temperature
?. [Purification of the Ligation Reaction]
Briefly spin the tube in a microcentrifuge to collect any sample on the sides of the tube
?. [Purification of the Ligation Reaction]
Place the tube on a magnetic rack for ~5 minutes to separate beads from the supernatant
?. [Purification of the Ligation Reaction]
Once the solution is clear, carefully remove and discard the supernatant without either removing or touching the beads
Be careful with the beads, they retain the target DNA
?. [Purification of the Ligation Reaction]
Add 200 μL of 80% ethanol to the tube while in the magnetic rack
?. [Purification of the Ligation Reaction]
Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant
?. [Purification of the Ligation Reaction]
Repeat from Step 102.8 once for a total of 2 washing steps
?. [Purification of the Ligation Reaction]
Briefly spin the tubes and put the tubes back on the magnetic rack
?. [Purification of the Ligation Reaction]
Remove the residual ethanol pipetting and air dry the beads for up to 5 minutes while the tube is on the    magnetic rack with lid open
Do not over-dry the beads. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry
?. [Purification of the Ligation Reaction]
Remove the tube from the magnetic rack
?. [Purification of the Ligation Reaction]
Add 17μL of of 0.1X TE Buffer (provided) to elute the DNA from the beads
?. [Purification of the Ligation Reaction]
Mix well on a vortex mixer
?. [Purification of the Ligation Reaction]
Quickly spin the tube and incubate for 2 minutes at
0 Room temperature
?. [Purification of the Ligation Reaction]
Place the tube on the magnetic rack for ~5 minutes, until the solution is clear
?. [Purification of the Ligation Reaction]
Remove 15μL of the supernatant and transfer to a clean 200µL nuclease free PCR tube without disturbing the beads pellet
If you need to stop, this is a safe stop point. Samples can be stored at -20°C
?. [Illumina library preparation]
Place the tube from step 102.18 on ice and assemble the following reaction:
The following PCR Enrichment was performed specifically using NEBNext Multiplex Oligos for Illumina (Index Primers set 1) (NEB #E7335S) with the oligos concentration of 10 µM. The PCR cycles are recommended using an initial RNA concentration of 100 ng; limit the PCR cycles to avoid overamplification with a secondary unwanted peak (~1000 bp) which will appear on the Bioanalyzer
The Universal PCR Primer should be added to all the samples while the indexing should be unique for each sample. See appendix 6    for an example of assembling reaction
?. [Illumina library preparation]
Place the sample in a thermocycler, upload and start program #7
For program #7 see appendix 5
?. Once the PCR amplification is ended proceed to the last Purification step
?. [Purification of the PCR Reaction]
Resuspend the NEBNext Sample Purification Beads by vortexing until the slurry is homogeneous
?. [Purification of the PCR Reaction]
Add 45μL (0.9X) of resuspended beads to the tubes from step 100 (PCR reaction, ~50μL)
?. [Purification of the PCR Reaction]
Incubate for 5 minutes at
0 Room temperature
?. [Purification of the PCR Reaction]
Mix well on a vortex mixer
?. [Purification of the PCR Reaction]
Briefly spin the tube in a microcentrifuge to collect any sample on the sides of the tube
?. [Purification of the PCR Reaction]
Place the tube on a magnetic rack for ~5 minutes to separate beads from the supernatant
?. [Purification of the PCR Reaction]
Once the solution is clear, carefully remove and discard the supernatant without either removing or touching the beads
Be careful with the beads, they retain the target DNA
?. [Purification of the PCR Reaction]
Add 200μL of 80% ethanol to the tubewhile in the magnetic rack
?. [Purification of the PCR Reaction]
Incubate at   for 30 seconds, and then carefully remove and discard the supernatant
0 Room temperature
?. [Purification of the PCR Reaction]
Repeat from Step 105.8 once for a total of 2 washing steps
?. [Purification of the PCR Reaction]
Briefly spin the tubes and put the tubes back on the magnetic rack
?. [Purification of the PCR Reaction]
Remove the residual ethanol pipetting and air dry the beads for up to 5 minutes while the tube is on the magnetic rack with lid open
Do not over-dry the beads. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack they are too dry
?. [Purification of the PCR Reaction]
Remove the tube from the magnetic rack
?. [Purification of the PCR Reaction]
Add 23μL of 0.1X TE Buffer (provided) to elute the DNA from the beads
?. [Purification of the PCR Reaction]
Mix well on a vortex mixer
?. [Purification of the PCR Reaction]
Quickly spin the tube and incubate for 2 minutes at
0 Room temperature
?. [Purification of the PCR Reaction]
Remove 20μL of the supernatant and transfer to a clean 200µL nuclease free PCR tube without disturbing the beads pellet
?. [Purification of the PCR Reaction]
Determine concentration of DNA (Step 105.17) using Qubit. Use 1 µL
?. [Purification of the PCR Reaction]
Store the DNA at -20°C or proceed to the library quality assessment on Bioanalyzer DNA chip
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Allow all reagents to equilibrate to room temperature for 30 minutes before use and protect dye and dye mixture from light
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Set up the priming station for Bioanalyzer® chip adjusting the base plate in C position and sliding the syringe clip up to the bottom positionPreparing the Gel-Dye Mix
Be sure of the clip position. Different position will lead to a negative output
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Add 15 μL of High Sensitivity DNA dye concentrate (blue ) to a High Sensitivity DNA gel matrix vial (red )
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Vortex solution well and spin down. Transfer to spin filter
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Centrifuge at 2240 g ± 20 % for 15 min. Store at 4 °C. Chip loading
Protect solution from light. Use prepared gel-dye mix within 6 weeks of preparation
Always insert the pipette tip to the bottom of the well when dispensing the liquid. Placing the pipette at the edge of the well may lead to poor results
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Place a new DNA chip on the chip priming station
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Pipette 9 μL of gel-dye mix in the well marked
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Make sure that the plunger is positioned at 1 mL and then close the chip priming station
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Press plunger until it is held by the clip
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Wait for exactly 60 seconds then release clip
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Wait for 5 seconds and slowly pull back plunger to 1 mL position
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Open the chip priming station and pipette 9 μL of gel-dye mix in the wells marked with
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Pipette 5 μL of DNA marker (green cap vial ) in all 11 sample wells and in the well marked
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Pipette 1 μL of High Sensitivity DNA ladder (yellow cap vial ) in the well marked
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Pipette 1μL of sample in each of the 11 sample wells. Pipette 1μL of DNA Marker  (green cap vial) in each unused sample well
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Positionate the chip horizontally in the IKA vortexer and vortex for 1 min at 2400rpm
DO NOT VORTEX FOR MORE THAN 1 MINUTE AND MORE THAN 2400rpm
?. [Library Average Fragment Length Assessment on an Agilent Bioanalyzer DNA Chip]
Place the chip in the Agilent 2100 Bioanalyzer and run the instrument within 5 min
Set up the Bioanalyzer instrument prior loading the chip. Keeping loaded gel for more than 5 minutes will allow the liquid in it to evaporate leading to a poor or non-acceptable analysis.For a clear overview of the DNA Average Fragment Length electropherogram output from the bioanalyzer see appendix 7
?. [qPCR quantification of the library prepared for Illumina sequencing]
Place all the reagents of the KAPA Library thawing on ice
If the kit is used for the first time, add the Primer Premix (10X) (1 mL) to the bottle of KAPA SYBR® FAST qPCR Master Mix (2X) (5 mL). Mix thoroughly using a vortex mixer
?. [qPCR quantification of the library prepared for Illumina sequencing]
Prepare the appropriate library dilutions (using DNA dilution buffer). Depending on the expected concentration of the library, 1:1,000 – 1:100,000 dilutions may be appropriate
All DNA Standards and library dilutions have to be assayed in triplicate.
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							Each sample concentration should fall within the dynamic range of given standards.Check appendix 8    to see examples of dilution calculations
?. [qPCR quantification of the library prepared for Illumina sequencing]
Setup the following reaction on ice using 96-well 0.2 ml PCR Plates
?. [qPCR quantification of the library prepared for Illumina sequencing]
Perform qPCR with the following cycling protocol:
?. [qPCR quantification of the library prepared for Illumina sequencing]
Perform the results analysis to check the library quantification
KAPABIOSYSTEMS provides a spreadsheet premade designed for the analysis of NGS library quantification data generated with the KAPA Library Quantification Kit for Illumina®. You can download it at the following link:https://www.google.it/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=2ahUKEwjAyPrV2s_hAhWxlYsKHW-mB9gQFjAAegQIARAC&url=https%3A%2F%2Fwww.kapabiosystems.com%2Fdocument%2Fkapa-library-quantification-data-analysis-template%2F%3Fdl%3D1&usg=AOvVaw0cKEl4Os4HF1F8a
?. [qPCR quantification of the library prepared for Illumina sequencing]
Normalize all the DNA samples to the lowest amount of DNA
?. [qPCR quantification of the library prepared for Illumina sequencing]
Pool the samples for the Illumina sequencing
?. [qPCR quantification of the library prepared for Illumina sequencing]
Store the samples at -80°C
?. [Water samples filtration]
Assemble the filtration system and place the 0.22 µm MF-membranes filters using RNase free forceps
?. [Appendix 1]
G2 test“G2 DNA/RNA Enhancer is convenient to use, when optimal DNA and or RNA extraction yield is required from especially clay. The primary function of G2 DNA/RNA Enhancer is to relieve inhibitory DNA-clay particle formations. G2 DNA/RNA Enhancer should be used in combination with either standardized extraction methods or commercial kits intended for DNA & RNA extraction from soil and clay” (Ampliqon).
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For this test were processed 500 mL and 1 mg of debris/soil for each sample with the addition of G2. According to protocol were added 2 mL of G2, to check the effect on the RNA extraction. A control without G2 was used.
?. [Appendix 2]
Relative Centrifugal Force (RCF) is the term used to describe the amount of accelerative force applied to a sample in a centrifuge. RCF is measured in multiples of the standard acceleration due to gravity at the Earth's surface (x g). This is why RCF and “x g” are used interchangeably in centrifugation protocols.
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							RCF or G = 1.12 x Radius (mm) x (rpm/1000)2
?. [Appendix 3]
Preparing the RNA Ladder
?. [Appendix 3]
Spin the ladder down and pipette in an RNase-free vial
?. [Appendix 3]
Heat denature the ladder for 2 min at
70 °C
?. [Appendix 3]
Immediately cool the vial on ice
?. [Appendix 3]
Add 90μL of RNase-free water and mix thoroughly
?. [Appendix 3]
Prepare aliquots in recommended 0.5 mL RNase-free vials with the required amount for typical daily use
?. [Appendix 3]
Store aliquots at  . After initial heat denaturation, the frozen aliquots should not require repeated heat denaturation
-80 °C
?. [Appendix 3]
Before use, thaw ladder aliquots on ice
Avoid extensive warming
?. [Appendix 3]
Preparing the Gel
?. [Appendix 3]
Pipette 550 μL of RNA gel matrix (red ) into a spin filter
?. [Appendix 3]
Centrifuge at 1500g ± 20 % for 10 min at
0 Room temperature
?. [Appendix 3]
Aliquot 65μL filtered gel into 0.5 mL RNase-free microcentrifuge tubes.Use filtered gel within 4 weeks. Store at  .
4 °C
?. [Appendix 4]
In the following section can be understood how the bioanalyzer works and the regions evaluated to check the final quality score
?. [Appendix 5]
PCR Tables
?. [Appendix 6]
Examples of how to perform PCR enrichment (Indexing) with 4 samples:
The only difference in the mixture is the Indexing choice (Highlighted in Bold). NebNext Multiplex Oligoes for Illumina (Index Primers Set 1) contains 12 index primers and they can all be used following the previous example for 12 samples.
?. [Appendix 7]
Assessment of DNA library fragment length average
If a peak at ~ 80 bp (primers) or 128 bp (adaptor-dimer) is visible in the Bioanalyzer traces; Bring up the sample volume (from Step 104.18) to 50 μl with 0.1X TE buffer and repeat the SPRIselect Bead or NEBNext Sample Purification Bead Cleanup Step (Section 105.1). 
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							A dilution may be necessary for running on a Bioanalyzer High Sensitivity DNA Chip
?. [Appendix 8]
qPCR dilutions calculationPrepare the appropriate library dilutions (using DNA dilution buffer). Depending on the expected concentration of the library, 1:1,000 – 1:100,000 dilutions may be appropriate. It is recommended to perform a ten-fold serial dilution to obtain a more reliable dilution.Follow the empiric calculation:1 ng/µL from the library with a 300 bp length it’s equal to say 3.5 nM in concentrationSo, 1ng/µL = 3.5 nM in concentrationPractical calculation:If we have a sample with 20 ng/µL from the library, multiply 20 ng/µL for 3.5 (conversion value) :20 ng/µL x 3.5 = 70 nMThe value in µM has to be transformed in pM and diluted a proper number of times to fall in the following standard table provided by the KAPABiosynthesis.70 nM = 70.000 pMPossibly we should dilute the samples to fall between standard 3 and 5.70.000 pM has to be diluted at least 100.000 times in order to obtain a final concentration of 0.7 pM. In this case an additional 10-fold dilution of each sample is recommended.