TITLE: Protocol for use with NEBNext® Small RNA Library Prep Set for Illumina® (E7300, E7580, E7560, E7330)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The NEBNext Multiplex Small RNA Library Prep Set for Illumina contains the adaptors, primers, enzymes and buffers required to convert small RNAs into indexed libraries for next generation sequencing on the Illumina platform. The novel workflow has been optimized to minimized adaptor dimers, while producing high-yield, high-diversity libraries. </div><div class = "text-block"><span>The unique workflow of the NEBNext</span><span style = "vertical-align:super;">®</span><span> Small RNA library prep kits addresses the challenge of minimization of adaptor-dimers while achieving production of high-yield, diverse multiplex libraries in a simple protocol. </span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Minimized adaptor-dimer contamination</li><li style = "counter-reset:ol0;list-style-type:disc;">High yields</li><li style = "counter-reset:ol0;list-style-type:disc;">Input RNA can be Total RNA</li><li style = "counter-reset:ol0;list-style-type:disc;">Suitable for methylated small RNAs (e.g., piRNAs) as well as unmethylated small RNAs</li></ul></div><div class = "text-block"><span style = "font-weight:bold;">Multiplex Small RNA Library Prep Workflow </span></div><div class = "text-block">This kit includes a novel protocol that results in higher yields and lower adaptor-dimer contamination.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/77wfw26.jpg" /></div></div>

[STEPS]
?. [Ligate the 3´ SR Adaptor]
Mix the following components in a sterile nuclease-free PCR tube. It is ok to premix the reagents. Use immediately.  AB1Input RNA1-6 µl2(green) 3 ́ SR Adaptor for Illumina1 µl3Nuclease-Free Watervariable4Total volume7 µl
For total RNA inputs of 100 ng, dilute the (green) 3´ SR Adaptor for Illumina 1:2 (For example: 1 µl of 3´ SR adaptor and 1 µl nuclease-free water) in nuclease-free water. For total RNA inputs closer to 1 µg, do not further dilute the adaptor. Adaptor dilutions may need to be optimized further.
?. [Ligate the 3´ SR Adaptor]
Incubate in a preheated thermal cycler for  at . Transfer tube to ice.
?. [Ligate the 3´ SR Adaptor]
Add and mix the following components. It is ok to premix the reagents. Use immediately.   AB1(green) 3 ́ Ligation Reaction Buffer (2X)10 µl2(green) 3 ́ Ligation Enzyme Mix3 µl3Total volume20 µl
?. [Ligate the 3´ SR Adaptor]
Incubate for  at   in a thermal cycler.
Longer incubation times and reduced temperatures ( ;  ) increase ligation efficiency of methylated RNAs such as piwi-interacting RNAs (piRNAs) (if present in the sample). However, some concatamerization products might be formed.
?. [Hybridize the Reverse Transcription Primer]
This section is important to prevent adaptor-dimer formation. The SR RT Primer hybridizes to the excess of 3´ SR Adaptor (that remains free after the 3´ ligation reaction) and transforms the single stranded DNA adaptor into a double-stranded DNA molecule. dsDNAs are not substrates for ligation mediated by T4 RNA Ligase 1 and therefore do not ligate to the 5´ SR Adaptor in the subsequent ligation step.
For total RNA inputs of 100 ng, dilute the (pink) SR RT Primer for Illumina 1:2 in nuclease free water. For total RNA inputs closer to 1 µg do not dilute the primer. Depending on the small RNA quantity and quality of your sample additional dilution optimization may be required.
?. [Hybridize the Reverse Transcription Primer]
Add and mix the following components to the ligation mixture from Step 4 and mix well. It is ok to premix the reagents.  AB1Nuclease-Free Water4.5 µl2(pink) SR RT Primer for Illumina1 µl3Total volume now should be25.5 µl
?. [Hybridize the Reverse Transcription Primer]
Place in a thermocycler with heated lid set to > and run the following program:5 minutes at 15 minutes at 15 minutes at Hold at
?. [Ligate the 5´ SR Adaptor]
With  remaining, resuspend the (yellow) 5´ SR adaptor in .
[nuclease free water]
For total RNA inputs closer to 100 ng, additionally dilute the (yellow) 5´ SR Adaptor for Illumina 1:2 in nuclease free water. For total RNA inputs closer to 1 µg do not dilute the adaptor further.
?. [Ligate the 5´ SR Adaptor]
Aliquot the (yellow) 5´ SR Adaptor into a separate, nuclease-free 200 µl PCR tube, for the number of samples in the experiment plus an excess of 10%.
?. [Ligate the 5´ SR Adaptor]
Incubate the adaptor in the thermal cycler at for  and then immediately place the tube on ice. Keep the tube on ice and use the denatured adaptor within  of denaturation.
Store the remaining resuspended 5´ SR adaptor at  . Denature aliquots before use. Please minimize freeze/thaw cycles. If only a few libraries are to be made at a time, the 5´ SR adaptor could be aliquoted.
?. [Ligate the 5´ SR Adaptor]
Add and mix the following components to the ligation mixture from Step 7 and mix well. Do not premix reagents.  AB1(yellow) 5 ́ SR Adaptor for Illumina (denatured)1 µl2(yellow) 5 ́ Ligation Reaction Buffer (10X)1 µl3(yellow) 5 ́ Ligation Enzyme Mix2.5 µl4Total volume30 µl
?. [Ligate the 5´ SR Adaptor]
Incubate for  at  in a thermal cycler.
?. [Perform Reverse Transcription]
Mix the following components in a sterile, nuclease-free tube. It is ok to premix the reagents. Use immediately.  AB1Adaptor Ligated RNA from Step 1230 µl2(red) First Strand Synthesis Reaction Buffer8 µl3(red) Murine RNase Inhibitor1 µl4(red) ProtoScript IIReverse Transcriptase1 µl5Total volume40 µl
?. [Perform Reverse Transcription]
Incubate for  at .
?. [Perform Reverse Transcription]
Immediately proceed to PCR amplifcation.
Safe Stopping Point: If you do not plan to proceed immediately to PCR amplifcation, then heat inactivate the RT reaction at   for  . Samples can be safely stored at   to
?. [Perform PCR Amplifcation]
Add and mix the following components to the RT reaction mix from Step 14 and mix well:  AB1(blue) LongAmp Taq 2X Master Mix50 µl2(blue) SR Primer for Illumina2.5 µl3(blue) Index (X) Primer*2.5 µl4Nuclease free water5 µl5Total volume now should be100 µl
*  Note: The NEBNext Multiplex Small RNA Library Prep Set for Illumina Set 1 contains 1–12 PCR primers, set 2 contains 23-24 PCR primers, kit index primers 1-48 PCR primer, each with a different index. For each reaction, only one of the 12 PCR primer indices is used during the PCR step.
?. [Perform PCR Amplifcation]
PCR Cycling conditions:  ABCD1CYCLE STEPTEMPTIMECYCLES2Initial Denaturation94°C30 sec13Denaturation94°C15 sec12-15*4Annealing62°C30 sec5Extension70°C15 sec6Final Extension70°C5 min17Hold4°C∞*Amplifcation conditions may vary based on RNA input amount, tissue, and species. This protocol was optimized using 1 µg of total RNA from human brain and 12 PCR cycles. The number of PCR cycles may need to be adjusted if clear and distinct bands are not observed in the gel image. For 100 ng total RNA input run 15 cycles of PCR. For samples containing high amounts of small RNA, less than 12 cycles may be appropriate.
Safe Stopping Point: It is safe to store the library at   after PCR. Avoid leaving the sample at   overnight if possible.
?. [Quality Control Check and Size Selection]
There are several different methods for performing size selection. It is recommended to choose the appropriate method based on the QC check of the library using the Bioanalyzer. Size selection using AMPure XP Beads does not remove small fragments. If you perform the QC check and your sample contains Adaptor dimer (127 bp peak) or excess primers (70-80 bp) it is recommended to use gel or Pippin Prep for size selection. Please selcet either case Option A, Option B, or Option C below. Option A: QC Check and Size Selection using 6% PolyAcrylamide GelOption B: QC Check and Size Selection Using Pippin PrepSize selection of the Small RNA library (147 bp) can done on Pippin Prep instrument using the 3% Agarose, dye free gel with internal standards (Sage Science # CDP3010).Option C: QC Check and Size Selection using AMPure XP BeadsBead size selection is only recommended for samples showing no primer dimer and no adaptor dimer on Bioanalyzer. It will be suitable to remove peaks > 150 bp. If fragments larger than 150 bp are abundant, two rounds of bead size selection may be necessary to completely eliminate the high molecular weight fragments.
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Purify the PCR amplified cDNA construct () using a Monarch PCR & DNA Kit.IMPORTANT: Use the 7:1 ratio of binding buffer:sample.Discard the flow through after each centrifugation step.
100 µl
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Elute amplified DNA in .
[Nuclease-free Water]
Safe Stopping Point: It is safe to store the library at  .
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Load  on the Bioanalyzer using a DNA 1000 chip according to the manufacturer's instructions (Figure 1).
[purified PCR reaction]
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Mix the purified PCR product () with .
25 µl
[Gel Loading Dye, Blue (6X)]
Vortex the Gel Loading Dye, Blue throughly to mix well before using.
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Load  in one well on the 6% PAGE 10-well gel.
[Quick-Load pBR322 DNA-MspI Digest]
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Load two wells with each of mixed amplified cDNA construct and loading dye on the 6% PAGE 10-well gel.
15 µl
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Run the gel for  at 120 V or until the blue dye reaches the bottom of the gel. Do not let the blue dye exit the gel.
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Remove the gel from the apparatus and stain the gel with SYBR Gold nucleic acid gel stain in a clean container for –minutes and view the gel on a UV transiluminator (Figure 2).
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
The 140 and 150 nucleotide bands correspond to adapter-ligated constructs derived from the 21 and 30 nucleotide RNA fragments, respectively. For miRNAs, isolate the bands corresponding to ~140 bp. For piRNAs, isolate the band corresponding to ~150 bp. For other small RNA, the band size may be different.
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Place the two gel slices from the same sample in one 1.5 ml tube and crush the gel slices with the RNase-free Disposable Pellet Pestles and then soak in .
[DNA Gel Elution buffer (1X)]
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Rotate end-to-end for at least at room temperature.
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Transfer the eluate and the gel debris to the top of a gel filtration column (for example: Corning®, Costar®, Spin-X® Centrifuge Tube Filters (Cellulose Acetate Filters) (Sigma Aldrich #CLS8162).
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Centrifuge the filter for  at > 13,200 rpm.
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Recover eluate and add ,  and .
[ Linear Acrylamide]
[3M sodium acetate, pH 5.5]
[100% ethanol]
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Vortex well.
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Precipitate in a dry ice/methanol bath or at  for at least .
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Spin in a microcentrifuge @ > 14,000 x g for  at .
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Remove the supernatant taking care not to disturb the pellet.
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Wash the pellet with 80% ethanol by vortexing vigorously.
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Spin in a microcentrifuge @ > 14,000 x g for  at .
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Air dry pellet for up to  at room temperature to remove residual ethanol.
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Resuspend pellet in .
[TE Buffer]
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Load  of the size selected purified library on a 2100 Bioanalyzer using a DNA 1000 or High Sensitivity DNA chip according to the manufacturer's instructions (Figure 3).
1 µl
?. [QC Check and Size Selection using 6% PolyAcrylamide Gel]
Check the size, purity, and concentration of the sample.
?. [QC Check and Size Selection Using Pippin Prep]
Purify the PCR amplified cDNA construct (100 μl) using a Monarch PCR & DNA Cleanup Kit.IMPORTANT: Use the 7:1 ratio of binding buffer:sample.Discard the flow through after each centrifugation step.
?. [QC Check and Size Selection Using Pippin Prep]
Elute amplified DNA in .
[nuclease-free water]
Safe Stopping Point: It is safe to store the library at   after PCR cleanup.
?. [QC Check and Size Selection Using Pippin Prep]
It is recommended to QC your library before performing size selection:Load of the purified PCR reaction on the Bioanalyzer using a DNA 1000 chip according to the manufacturer's instructions (Figure 1). . miRNA library should appear as a peak at 147 bp peak (that correspond for 21 nucleotide insert).
1 µl
?. [QC Check and Size Selection Using Pippin Prep]
Program the protocol for size selection on Pippin Prep Instrument as described in the following steps:In the Pippin Prep software, go to the Protocol Editor Tab.
?. [QC Check and Size Selection Using Pippin Prep]
Click “Cassette” folder, and select “3% DF Marker P”.
?. [QC Check and Size Selection Using Pippin Prep]
Select the collection mode as “Range” and enter the size selection parameters as follow: BP start (105) and the BP end (155). BP Range Flag should indicate “broad”.
This protocol is optimized to select for 147–149 bp peak. When targeting other small RNA these settings may have to be adjusted.
?. [QC Check and Size Selection Using Pippin Prep]
Click the “Use of Internal Standards” button.
?. [QC Check and Size Selection Using Pippin Prep]
Make sure the “Ref Lane” values match the lane numbers.
?. [QC Check and Size Selection Using Pippin Prep]
Press “Save As” and name and save the protocol.
?. [QC Check and Size Selection Using Pippin Prep]
Prepare sample for size selection as described in the follwing steps: Bring loading solution to room temperature.
?. [QC Check and Size Selection Using Pippin Prep]
For each sample, combine  with .
[sample]
[DNA marker P (labeled P)]
?. [QC Check and Size Selection Using Pippin Prep]
Mix samples thoroughly (vortex mixer). Briefly centrifuge to collect.
?. [QC Check and Size Selection Using Pippin Prep]
Load (DNA plus marker) on one well of the 3% agarose cassette.
40 µl
?. [QC Check and Size Selection Using Pippin Prep]
Run the program with the settings indicated above.
?. [QC Check and Size Selection Using Pippin Prep]
After sample has been eluted, collect 40 µl sample from elution well. Run  in a Bioanalzyer using the high sensitivity chip.
1 µl
If the Ethidium Bromide free cassettes was used, no purification is required before running sample on the bioanalyzer.
?. [QC Check and Size Selection using AMPure XP Beads]
Purify the PCR amplified cDNA construct () using a Monarch PCR & DNA Kit.IMPORTANT: Use the 7:1 ratio of binding buffer:sample. Discard the flow through after each centrifugation step.
100 µl
?. [QC Check and Size Selection using AMPure XP Beads]
Elute amplified DNA in .
[Nuclease-free Water]
Safe Stopping Point: It is safe to store the library at   after PCR cleanup.
?. [QC Check and Size Selection using AMPure XP Beads]
Load  on the Bioanalyzer using a DNA 1000 chip according to the manufacturer's instructions (Figure 1).
[purified PCR reaction]
?. [QC Check and Size Selection using AMPure XP Beads]
To the purified PCR reaction (), add (1.3X) of resuspended AMPure XP beads and mix well on a vortex mixer or by pipetting up and down at least 10 times.
25 µl
32 µl
?. [QC Check and Size Selection using AMPure XP Beads]
Incubate for  at room temperature.
?. [QC Check and Size Selection using AMPure XP Beads]
Place the tube on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about ), carefully transfer the supernatant () to a new tube (Caution: do not discard the supernatant). Discard beads that contain the large DNA fragments.
57 µl
?. [QC Check and Size Selection using AMPure XP Beads]
Add  (3.7X) of resuspended AMPure XP beads to the supernatant (), mix well and incubate for at room temperature.
92 µl
57 µl
?. [QC Check and Size Selection using AMPure XP Beads]
Place the tube on an appropriate magnetic stand to separate beads from supernatant. After the solution is clear (about ), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard beads).
?. [QC Check and Size Selection using AMPure XP Beads]
Add  to the tube while in the magnetic stand. Incubate at room temperature for , and then carefully remove and discard the supernatant. (1/2)
[ freshly prepared 80% ethanol]
?. [QC Check and Size Selection using AMPure XP Beads]
Add  to the tube while in the magnetic stand. Incubate at room temperature for , and then carefully remove and discard the supernatant. (2/2)
[ freshly prepared 80% ethanol]
?. [QC Check and Size Selection using AMPure XP Beads]
Briefly spin the tube, and put the tube back in the magnetic stand.
?. [QC Check and Size Selection using AMPure XP Beads]
Completely remove the residual ethanol, and air dry beads for up to  while the tube is on the magnetic stand with lid open.Caution: Do not overdry the beads, which may result in lower recovery of the DNA target. Elute the sample 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.
?. [QC Check and Size Selection using AMPure XP Beads]
Elute the DNA target from the beads with . Mix well on a vortex mixer or by pipetting up and down, incubate for  and put the tube in the magnetic stand until the solution is clear.
[nuclease-free water]
?. [QC Check and Size Selection using AMPure XP Beads]
Transfer the supernatant to a clean PCR tube.
?. [QC Check and Size Selection using AMPure XP Beads]
Run on the Bioanalyzer High Sensitivity chip. Check peak distribution and concentration of the small RNA library.
1 µl