TITLE: Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of input amounts of RNA into high quality directional (strand-specific) libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow has minimal hands-on time and is compatible with poly(A) mRNA enrichment and rRNA depletion methods.</div></div>

[STEPS]
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Dilute the total RNA with nuclease-free water to a final volume of  in a nuclease-free 0.2 ml PCR tube and keep on ice.
50 µl
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
To wash the Oligo dT Beads, add the following to a 1.5 ml nuclease-free tube. If preparing multiple libraries, beads for up to 10 samples can be added to a single 1.5 ml tube for subsequent washes (use magnet NEB #S1506 for 1.5 ml tubes). The purpose of this step is to bring the beads from the storage buffer into the binding buffer. The 2X Binding Buffer does not have to be diluted for this step.   AB1ComponentVolume Per One Library2Oligo dT Beads d(T)2520 µl3RNA Binding Buffer100 µl4Total Volume120 µl
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Wash the beads by pipetting up and down 6 times.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Place the tube on the magnet and incubate at room temperature until the solution is clear (~).
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove the tube from the magnetic rack.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Add  RNA Binding Buffer (2X) to the beads and wash by pipetting up and down six times. If preparing multiple libraries, add  RNA Binding Buffer (2X) per sample. The Binding Buffer does not have to be diluted.
100 µl
100 µl
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Place the tubes on the magnet and incubate at room temperature until the solution is clear (~).
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove and discard the supernatant from the tube. Take care not to disturb the beads.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Add 50 μl beads to each RNA sample from Step 2. Mix thoroughly by pipetting up and down 6 times.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Place the tube in a thermocycler and close the lid. Heat the sample at  for  and cool to  with the heated lid set at ≥  to denature the RNA and facilitate binding of the mRNA to the beads.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove the tube from the thermocycler when the temperature reaches .
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Place the tube on the magnetic rack at room temperature until the solution is clear (~).
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove and discard all of the supernatant. Take care not to disturb the beads.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove the tube from the magnetic rack.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Wash the beads by adding  of Wash Buffer to the tube to remove unbound RNA. Gently pipette the entire volume up and down 6 times to mix thoroughly.
200 µl
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove and discard all of the supernatant from the tube. Take care not to disturb the beads.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Place the tube on the magnetic rack at room temperature until the solution is clear (~).
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove the tube from the magnetic rack.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Repeat steps 20–23.
[Repeat]
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Add 50 μl of Tris Buffer (provided in NEB #E7490 kit) to each tube. Gently pipette up and down 6 times to mix thoroughly.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove the tube from the thermocycler when the temperature reaches .
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Add 50 μl of RNA Binding Buffer (2X) to the sample to allow the mRNA to re-bind to the beads. Mix thoroughly by gently pipetting up and down six times.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Incubate the tube at room temperature for .
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Place the tube on the magnetic rack at room temperature until the solution is clear (~).
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove and discard the supernatant from the tube. Take care not to disturb the beads.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove the tube from the magnetic rack.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Wash the beads by adding  of Wash Buffer. Gently pipette the entire volume up and down 6 times to mix thoroughly.
200 µl
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Spin down the tube briefly to collect the liquid from the wall and lid of the tube.
It is important to spin down the tube to prevent carryover of the Wash Buffer in subsequent steps.
?. [Probe Hybridization to RNA]
Mix thoroughly by pipetting up and down 10 times.
Keep the mix on ice until mRNA is purified. It will be used in Step 38 and 41.
?. [Probe Hybridization to RNA]
Prepare the First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) in a nuclease-free microcentrifuge tube as follows:   AB1ComponentVolume2NEBNext First Strand Synthesis Reaction Buffer8 µl3NEBNext Random Primers2 µl4Nuclease-free Water10 µl5Total Volume20 µlYou can prepare the first strand synthesis reaction buffer later in the protocol, but it is important that it is ready before the elution in step 38. The beads should not be allowed to dry out.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Add RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous. If preparing multiple libraries, add  RNA Binding Buffer (2X) per sample. This first binding step removes most of the non target RNA.
50 µl
50 µl
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Mix thoroughly by pipetting up and down 6 times. Place the tube on the bench and incubate at room temperature for  to allow the mRNA to bind to the beads.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Place the tube on the thermocycler. Close the lid and heat the samples at for, then cool to  with the heated lid set at ≥  to do the first elution of the mRNA from the beads.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Place the tube on the magnet at room temperature until the solution is clear (~).
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove and discard all of the supernatant from the tube. Take care not to disturb the beads that contains the mRNA.
It is important to remove all of the supernatant to successfully fragment the mRNA in the subsequent steps. Spin down the tube. Place the tube on the magnetic rack and with a 10 μl tip, remove all of the wash buffer. (Caution: Do not disturb beads that contain the mRNA). Avoid letting the beads dry out before adding elution buffer.
?. [First Strand cDNA Synthesis]
Assemble the first strand synthesis reaction on ice by adding the following components to the fragmented and primed RNA from Step 42:  AB1First Strand Synthesis ReactionVolume2Fragmented and Primed RNA (Step 36)10 µl3NEBNext Strand Specificity Reagent8 µl4NEBNext First Strand Synthesis Enzyme Mix2 µl5Total Volume20 µl
?. [First Strand cDNA Synthesis]
Mix thoroughly by pipetting up and down 10 times.
?. [First Strand cDNA Synthesis]
[ ! ] Incubate the sample in a preheated thermocycler with the heated lid set at ≥ 80°C as follows:Note: If you are following recommendations in Appendix A (Chapter 6), for libraries with longer inserts (> 200 bases), increase the incubation at  from 15 minutes to 50 minutes at Step 2 below.Step 1:  at Step 2:  at Step 3:  at Step 4: Hold at
?. [First Strand cDNA Synthesis]
Proceed directly to Second Strand cDNA Synthesis.
?. [Second Strand cDNA Synthesis]
Assemble the second strand cDNA synthesis reaction on ice by adding the following components into the first strand synthesis product from Step 46).  AB1Second&nbsp;Strand Synthesis ReactionVolume2First Strand Synthesis Product (Step 46)20 µl3NEBNext Second Strand Synthesis Reaction Buffer with dUTP (10X)8 µl4NEBNext Second Strand Synthesis Enzyme Mix4 µl5Nuclease-free Water48 µl6Total Volume80 µl
?. [Second Strand cDNA Synthesis]
Keeping the tube on ice, mix thoroughly by pipetting up and down at least 10 times.
?. [Second Strand cDNA Synthesis]
Incubate in a thermocycler for  at  with the heated lid set at  (or off).
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Add  (1.8X) of resuspended beads to the second strand synthesis reaction (~). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
144 µl
80 µl
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Incubate for  at room temperature.
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Briefly spin the tube in a microcentrifuge to collect any sample on the sides of the tube. Place the tube on a magnet to separate beads from the supernatant. After the solution is clear, carefully remove and discard the supernatant. Be careful not to disturb the beads, which contain DNA. Caution: Do not discard beads.
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Add  of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for , and then carefully remove and discard the supernatant.
200 µl
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Repeat Step 54 once for a total of 2 washing steps.
[Repeat Step]
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Air dry the beads for up to 5 minutes while the tube is on the magnetic rack with lid open.Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. 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 Using SPRIselect Beads or NEBNext Sample Purification Beads]
Remove the tube from the magnetic rack. Elute the DNA from the beads by adding  0.1X TE Buffer (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down at least 10 times. Quickly spin the tube and incubate for  at room temperature. Place the tube on the magnetic rack until the solution is clear.
53 µl
?. [Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads]
Remove  of the supernatant and transfer to a clean nuclease-free PCR tube.
50 µl
If you need to stop at this point in the protocol samples can be stored at –20°C.
?. [End Prep of cDNA Library]
Assemble the end prep reaction on ice by adding the following components to the second strand synthesis product from Step 58.  AB1End Prep ReactionVolume2Second Strand Synthesis Product (Step 58)50 µl3NEBNext Ultra II End Prep Reaction Buffer7 µl4NEBNext Ultra II End Prep Enzyme Mix3 µl5Total Volume60 µlIf a master mix is made, add  of master mix to  of cDNA for the End Prep reaction.
10 µl
50 µl
?. [End Prep of cDNA Library]
Set a 100 μl or 200 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 with performance.
?. [End Prep of cDNA Library]
Incubate the sample in a thermocycler with the heated lid set at ≥ as follows. at  at Hold at
?. [End Prep of cDNA Library]
Proceed immediately to Adaptor Ligation.
?. [Adaptor Ligation]
[ ! ] Dilute the NEBNext Adaptor* prior to setting up the ligation reaction in ice-cold Adaptor Dilution Buffer and keep the adaptor on ice.  AB1Total RNA InputDilution Required21,000 ng–250 ng5–fold dilution in Adaptor Dilution Buffer3249 ng–100 ng25–fold dilution in Adaptor Dilution Buffer499 ng–10 ng100–fold dilution in Adaptor Dilution Buffer*The NEBNext adaptor is provided in NEBNext oligos kit. NEB has several oligo kit options, which are supplied separately from the library prep kit.
?. [Adaptor Ligation]
Assemble the ligation reaction on ice by adding the following components, in the order given, to the end prep reaction product from Step 62.  AB1Ligation ReactionVolume2End Prepped DNA (Step 62)60 µl3Diluted Adaptor (Step 63)2.5 µl4NEBNext Ligation Enhancer1 µl5NEBNext Ultra II Ligation Master Mix30 µl6Total Volume93.5 µl
The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @  . We do not recommend premixing the Ligation Master Mix, Ligation Enhancer and adaptor prior to use in the Adaptor Ligation Step.
?. [Adaptor Ligation]
Set a 100 μl or 200 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.[ ! ] Caution: 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.
?. [Adaptor Ligation]
Incubate 15 minutes at 20°C in a thermocycler.
[Incubate]
?. [Adaptor Ligation]
Add  (blue) USER™ Enzyme to the ligation mixture from Step 60, resulting in total volume of
3 µl
96 µl
?. [Adaptor Ligation]
Mix well and incubate at  for with the heated lid set to ≥.
?. [Adaptor Ligation]
Proceed immediately to Purification of the Ligation Reaction.
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
[ ! ] Note: If you are selecting for libraries with larger insert size (> 200 nt) follow the size selection recommendations in Appendix A, Chapter 6. Add  (0.9X) resuspended SPRIselect Beads or NEBNext Sample Purification Beads and mix well on a vortex mixer or by pipetting up and down at least 10 times.
87 µl
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Incubate for  at room temperature.
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Quickly spin the tube in a microcentrifuge and place the tube on an appropriate magnetic rack to separate beads from the supernatant. After the solution is clear (~ ), discard the supernatant that contains unwanted fragments.Caution: Do not discard beads.
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Add  of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for , and then carefully remove and discard the supernatant.
200 µl
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Repeat Step 73 once for a total of 2 washing steps.
[Repeat Step]
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Briefly spin the tube, and put the tube back in the magnetic rack.
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Completely remove the residual ethanol, and air dry beads until the beads are dry for up to 5 minutes while the tube is on the magnetic rack with the lid open.[ ! ] Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. 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 Using SPRIselect Beads or NEBNext Sample Purification Beads]
Remove the tube from the magnetic rack. Elute DNA target from the beads by adding  0.1X TE (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down. Quickly spin the tube and incubate for  at room temperature. Put the tube in the magnet until the solution is clear.
17 µl
?. [Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads]
Without disturbing the bead pellet, transfer  of the supernatant to a clean PCR tube and proceed to PCR enrichment.
15 µl
If you need to stop at this point in the protocol samples can be stored at –20°C.
?. [PCR Enrichment of Adaptor Ligated DNA]
[ ! ] Check and verify that the concentration of your oligos is 10 μM on the label.[ ! ] Use Option A for any NEBNext oligos kit where index primers are supplied in tubes. These kits have the forward and reverse primers supplied in separate tubes.Use Option B for any NEBNext oligos kit where index primers are supplied in a 96-well plate format. These kits have the forward and reverse (i7 and i5) primers combined.
?. [PCR Enrichment of Adaptor Ligated DNA]
Set up the PCR reaction as described below based on the type of oligos (PCR primers) used.Option A: Forward and Reverse Primers Separate:  AB1ComponentVolume Per One Library2Adaptor Ligated DNA (Step 78)15 µl3NEBNext Ultra II Q5 Master Mix25 µl4Universal PCR Primer/i5 Primer*,**5 µl5Index (X) Primer/i7 Primer*,**5 µl6Total Volume50 µlOption B: Forward and Reverse Primers Combined:  AB1ComponentVolume Per One Library2Adaptor ligated DNA (Step 78)15 µl3NEBNext Ultra II Q5 Master Mix25 µl4Index (X)/Universal Primer Mix*10 µl5Total Volume50 µl* NEBNext Oligos must be purchased separately from the library prep kit. Refer to the corresponding NEBNext Oligo kit manual for determining valid barcode combinations.** Use only one i7 primer/ index primer per sample. Use only one i5 primer (or the universal primer for single index kits) per sample
?. [PCR Enrichment of Adaptor Ligated DNA]
Mix well by gently pipetting up and down 10 times. Quickly spin the tube in a microcentrifuge.
?. [PCR Enrichment of Adaptor Ligated DNA]
Place the tube on a thermocycler with the heated lid set to  and perform PCR amplification using the following PCR cycling conditions (refer to Table 82.A and Table 82.B):Table 82.A:  ABCD1Cycle StepTempTimeCycles2Initial&nbsp;Denaturation98°C30 seconds13Denaturation98°C10 seconds8–16*,**4Annealing/Extension65°C15 seconds5Extension72°C20 seconds6Final Extension72°C5 minutes17Hold4°C∞&nbsp;* The number of PCR cycles should be adjusted based on RNA input.** It is important to limit the number of PCR cycles to avoid overamplification. If overamplification occurs, a second peak ~ 1,000 bp will appear on the Bioanalyzer trace (See Figure 7.2 in Manual).Table 82.B: Recommended PCR cycles based on total RNA input amount:  AB1Total RNA InputRecommended PCR Cycles21,000 ng8–93100 ng12–13410 ng15–16Note:  PCR cycles are recommended based on high quality Universal Human Reference Total RNA. It may requireoptimization based on the sample quality to prevent PCR over-amplification.
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Add  (0.9X) of resuspended beads to the PCR reaction (~). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
45 µl
50 µl
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Incubate for  at room temperature.
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Quickly spin the tube in a microcentrifuge and place the tube on an appropriate magnetic rack to separate beads from the supernatant. After the solution is clear (~), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets. Caution: Do not discard beads.
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Add  of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for , and then carefully remove and discard the supernatant.
200 µl
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Repeat  once for a total of 2 washing steps.
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Air dry the beads for up to 5 minutes while the tube is on the magnetic rack with the lid open. Caution: Do not over-dry the beads. This may result in lower recovery of DNA target. 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 using SPRIselect Beads or NEBNext Sample Purification Beads]
Remove the tube from the magnetic rack. Elute the DNA target from the beads by adding  0.1X TE (provided) to the beads. Mix well on a vortex mixer or by pipetting up and down ten times. Quickly spin the tube in a microcentrifuge and incubate for  at room temperature. Place the tube in the magnetic rack until the solution is clear.
23 µl
?. [Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads]
Transfer  of the supernatant to a clean PCR tube, and store at –20°C.
20 µl
?. [Assess Library Quality on an Agilent Bioanalyzer DNA Chip]
Run  library on a DNA 1000 chip. If the library yield is too low to quantify on this chip, please run the samples on a DNA High Sensitivity chip. A dilution may be necessary for running on a Bioanalyzer High Sensitivity DNA Chip.
1 µl
?. [Assess Library Quality on an Agilent Bioanalyzer DNA Chip]
Check that the electropherogram shows a narrow distribution with a peak size approximately 300 bp.
Note: 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 85) to 50 μl with 0.1X TE buffer and repeat the SPRIselect Bead or NEBNext Sample Purification Bead Cleanup Step (Section "Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads").
?. [Assess Library Quality on an Agilent Bioanalyzer DNA Chip]
Figure 94: Example of RNA library size distribution on a Bioanalyzer.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Remove the tube from the magnetic rack.
[ ! ] The next step provides a fragmentation incubation time resulting in an RNA insert size of ~ 200 nt. For RNA insert sizes > 200 nt, refer to Chapter 6 (Appendix A) for recommended fragmentation times in Step 39.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
To elute the mRNA from the beads and fragment, add  of the First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) prepared in Step 2, pipette up and down six times to resuspend the beads.
11 µl
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Incubate the sample in a thermocycler with the heated lid set at  as follows:  at Hold at* *Immediately transfer the tube to ice for  as soon as it is cool enough to handle (~)
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Quickly spin down the tube in a microcentrifuge to collect the liquid from the sides of the tube and place on the magnet right away until the solution is clear (~1–2 minutes).
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Collect the fragmented mRNA by transferring  of the supernatant to a nuclease-free 0.2 ml PCR tube.
10 µl
If the supernatant volume recovered is less than 10 μl for any reason, bring the volume up to 10 μl by adding the First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) prepared in Step 2 and continue with the protocol.
Avoid transferring any of the magnetic beads.
?. [mRNA Isolation, Fragmentation and Priming Starting with Total RNA]
Place the tube on ice and proceed directly to First Strand cDNA Synthesis.