TITLE: Bulk RNA - 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: Aaron Horning, New England   Biolabs

[DESCRIPTION]
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.

[BEFORE_START]
Input Amount Requirement: 10 ng–1 μg DNA-free total RNA quantified by Qubit® Fluorometer and quality checked by Bioanalyzer. The protocol is optimized for approximately 200 bp RNA inserts. To generate libraries with longer RNA insert sizes, refer to Appendix A (Section 6) for recommended fragmentation times and size selection conditions.

Keep all the buffers on ice, unless otherwise indicated.

[GUIDELINES]
Section 1
RNA Sample Requirements

RNA Integrity:
Assess the quality of the Input RNA by running the RNA sample on an Agilent Bioanalyzer® RNA 6000 Nano/Pico Chip. For PolyA mRNA enrichment, high quality RNA with a RIN score > 7 is required.

RNA Sample Requirements:
The RNA sample should be free of salts (e.g. Mg2+, or guanidinium salts, divalent cation chelating agents (e.g. EDTA or EGTA) or organics (e.g. phenol or ethanol). RNA must be free of DNA. gDNA is a common contaminant from RNA preps. It may be carried over from the interphase of organic extractions or when the silica matrix of solid phase RNA purification methods is overloaded. If the total RNA sample may contain gDNA contamination, treat the sample with DNase I to remove all traces of DNA (DNase is not provided in this kit). After treatment with DNase I the enzyme should be removed from the sample. Any residual activity of the DNase I may degrade the oligos necessary for the enrichment. DNase I can be removed from the extraction using phenol/chloroform extraction and ethanol precipitation.

The protocol is optimized for approximately 200 bp RNA inserts. To generate libraries with longer RNA insert sizes, refer to Appendix A (Chapter 6) for recommended fragmentation times and size selection conditions. 

[STEPS]
SECTION: Probe Hybridization to RNA
4. Prepare the First Strand Synthesis Reaction Buffer and Random Primer Mix (2X) in a nuclease-free microcentrifuge tube as follows: 

 
You 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. 
SECTION: Probe Hybridization to RNA
5. Mix thoroughly by pipetting up and down 10 times.
 


SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
6. Dilute the total RNA with nuclease-free water to a final volume of 50 µL in a nuclease-free 0.2 ml PCR tube and keep on ice.
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
7. 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. 

 

SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
8. Wash the beads by pipetting up and down 6 times. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
9. Place the tube on the magnet and incubate at room temperature until the solution is clear (~2 min). 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
10. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
11. Remove the tube from the magnetic rack. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
12. Add 100 µL RNA Binding Buffer (2X) to the beads and wash by pipetting up and down six times. If preparing multiple libraries, add 100 µL RNA Binding Buffer (2X) per sample. The Binding Buffer does not have to be diluted.
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
13. Place the tubes on the magnet and incubate at room temperature until the solution is clear (~2 min). 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
14. Remove and discard the supernatant from the tube. Take care not to disturb the beads.
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
15. Add50 µL RNA Binding Buffer (2X) to the beads and mix by pipetting up and down until beads are homogenous. If preparing multiple libraries, add 50 µL RNA Binding Buffer (2X) per sample. This first binding step removes most of the non target RNA.
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
16. Add 50 μl beads to each RNA sample from Step 2. Mix thoroughly by pipetting up and down 6 times.
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
17. Place the tube in a thermocycler and close the lid. Heat the sample at 65 °C for 5 min and cool to 4 °C with the heated lid set at ≥ 75 °C to denature the RNA and facilitate binding of the mRNA to the beads.
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
18. Remove the tube from the thermocycler when the temperature reaches 4 °C.

SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
19. Mix thoroughly by pipetting up and down 6 times. Place the tube on the bench and incubate at room temperature for 5 min to allow the mRNA to bind to the beads.


SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
20. Place the tube on the magnetic rack at room temperature until the solution is clear (~2 min). 

SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
21. Remove and discard all of the supernatant. Take care not to disturb the beads. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
22. Remove the tube from the magnetic rack.
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
23. Wash the beads by adding 200 µL of Wash Buffer to the tube to remove unbound RNA. Gently pipette the entire volume up and down 6 times to mix thoroughly. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
24. Place the tube on the magnetic rack at room temperature until the solution is clear (~2 min).
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
25. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
26. Remove the tube from the magnetic rack. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
27. Repeat steps 20–23.
 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
28. Add 50 μl of Tris Buffer (provided in NEB #E7490 kit) to each tube. Gently pipette up and down 6 times to mix thoroughly.
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
29. Place the tube on the thermocycler. Close the lid and heat the samples at80 °C for2 min, then cool to 25 °C with the heated lid set at ≥ 90 °C to do the first elution of the mRNA from the beads.



SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
30. Remove the tube from the thermocycler when the temperature reaches 25 °C. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
31. 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. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
32. Incubate the tube at room temperature for 5 min. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
33. Place the tube on the magnetic rack at room temperature until the solution is clear (~2 min). 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
34. Remove and discard the supernatant from the tube. Take care not to disturb the beads. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
35. Remove the tube from the magnetic rack. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
36. Wash the beads by adding 200 µL of Wash Buffer. Gently pipette the entire volume up and down 6 times to mix thoroughly. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
37. Spin down the tube briefly to collect the liquid from the wall and lid of the tube. 
 

SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
38. Place the tube on the magnet at room temperature until the solution is clear (~2 min). 

SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
39. Remove and discard all of the supernatant from the tube. Take care not to disturb the beads that contains the mRNA. 

 

SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
40. Remove the tube from the magnetic rack. 
 



SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
41. To elute the mRNA from the beads and fragment, add 11.5 µL 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. 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
42. Incubate the sample in a thermocycler with the heated lid set at 105 °C as follows: 
15 min at 94 °C
Hold at4 °C* 

*Immediately transfer the tube to ice for 1 min as soon as it is cool enough to handle (~65 °C) 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
43. 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). 
SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
44. Collect the fragmented mRNA by transferring 10 µL of the supernatant to a nuclease-free 0.2 ml PCR tube. 
 

 


SECTION: mRNA Isolation (rRNA-depletion), Fragmentation and Priming Starting with Total RNA
45. Place the tube on ice and proceed directly to First Strand cDNA Synthesis. 
SECTION: First Strand cDNA Synthesis
46. Assemble the first strand synthesis reaction on ice by adding the following components to the fragmented and primed RNA from Step 42:
   First Strand Synthesis Reaction Volume   Fragmented and Primed RNA (Step 36) 10 µl   NEBNext Strand Specificity Reagent 8 µl   NEBNext First Strand Synthesis Enzyme Mix 2 µl   Total Volume 20 µl   
 
 

SECTION: First Strand cDNA Synthesis
47. Mix thoroughly by pipetting up and down 10 times.
SECTION: First Strand cDNA Synthesis
48. [ ! ] 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 42 °C from 15 minutes to 50 minutes at Step 2 below.
 
Step 1: 10 min at 25 °C
Step 2: 15 min at 42 °C
Step 3: 15 min at 70 °C
Step 4: Hold at 4 °C
SECTION: First Strand cDNA Synthesis
49. Proceed directly to Second Strand cDNA Synthesis.
SECTION: Second Strand cDNA Synthesis
50. Assemble the second strand cDNA synthesis reaction on ice by adding the following components into the first strand synthesis product from Step 46).
   Second Strand Synthesis Reaction Volume   First Strand Synthesis Product (Step 40) 20 µl   NEBNext Second Strand Synthesis Reaction Buffer with dUTP (10X) 8 µl   NEBNext Second Strand Synthesis Enzyme Mix 4 µl   Nuclease-free Water 48 µl   Total Volume 80 µl   
 
 
 
SECTION: Second Strand cDNA Synthesis
51. Keeping the tube on ice, mix thoroughly by pipetting up and down at least 10 times.
SECTION: Second Strand cDNA Synthesis
52. Incubate in a thermocycler for 60 min at 16 °C with the heated lid set at 40 °C (or off).
SECTION: Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads
53. Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.
 
SECTION: Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads
54. Add 144 µL (1.8X) of resuspended beads to the second strand synthesis reaction (~80 µL). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
SECTION: Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads
55. Incubate for 5 min at room temperature.
SECTION: Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads
56. 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.
SECTION: Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads
57. Add 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant.
 


SECTION: Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads
58. Repeat Step 54 once for a total of 2 washing steps.
 

SECTION: Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads
59. 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. 
SECTION: Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads
60. Remove the tube from the magnetic rack. Elute the DNA from the beads by adding 53 µL 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 2 min at room temperature. Place the tube on the magnetic rack until the solution is clear.
 

SECTION: Purification of Double-stranded cDNA Using SPRIselect Beads or NEBNext Sample Purification Beads
61. Remove 50 µL of the supernatant and transfer to a clean nuclease-free PCR tube.
 

SECTION: End Prep of cDNA Library
62. Assemble the end prep reaction on ice by adding the following components to the second strand synthesis product from Step 58.
   End Prep Reaction Volume   Second Strand Synthesis Product (Step 52) 50 µl   NEBNext Ultra II End Prep Reaction Buffer 7 µl   NEBNext Ultra II End Prep Enzyme Mix 3 µl   Total Volume 60 µl   
If a master mix is made, add 10 µL of master mix to 50 µL of cDNA for the End Prep reaction.
 
 


SECTION: End Prep of cDNA Library
63. 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.
 
SECTION: End Prep of cDNA Library
64. Incubate the sample in a thermocycler with the heated lid set at ≥ 75 °Cas follows.
30 min at 20 °C
30 min at 65 °C
Hold at4 °C
SECTION: End Prep of cDNA Library
65. Proceed immediately to Adaptor Ligation.
SECTION: Adaptor Ligation
66. [ ! ] Dilute the NEBNext Adaptor* prior to setting up the ligation reaction in ice-cold Adaptor Dilution Buffer and keep the adaptor on ice.
   Total RNA Input Dilution Required   1,000 ng–101 ng 5–fold dilution in Adaptor Dilution Buffer   100 ng–10 ng 25–fold dilution in Adaptor Dilution Buffer   5 ng 200–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.

SECTION: Adaptor Ligation
67. Assemble the ligation reaction on ice by adding the following components, in the order given, to the end prep reaction product from Step 62.
   Ligation Reaction Volume   End Prepped DNA (Step 56) 60 µl   Diluted Adaptor (Step 57) 2.5 µl   NEBNext Ligation Enhancer 1 µl   NEBNext Ultra II Ligation Master Mix 30 µl   Total Volume 93.5 µl   
 
 
 
SECTION: Adaptor Ligation
68. 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. 

SECTION: Adaptor Ligation
69. Incubate 15 minutes at 20°C in a thermocycler.
15 min
SECTION: Adaptor Ligation
70. Add 3 µL (blue) USER™ Enzyme to the ligation mixture from Step 60, resulting in total volume of 96.5 µL
 


SECTION: Adaptor Ligation
71. Mix well and incubate at 37 °C for 15 minwith the heated lid set to ≥45 °C.

SECTION: Adaptor Ligation
72. Proceed immediately to Purification of the Ligation Reaction.
SECTION: Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads
73. [ ! ] Note: If you are selecting for libraries with larger insert size (> 200 nt) follow the size selection recommendations in Appendix A, Chapter 6. 
 
Add 87 µL (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.
 


SECTION: Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads
74. Incubate for 10 min at room temperature.
SECTION: Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads
75. 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 (~ 5 min), discard the supernatant that contains unwanted fragments.

Caution: Do not discard beads. 
SECTION: Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads
76. Add 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant.
 


SECTION: Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads
77. Repeat Step 73 once for a total of 2 washing steps.
 

SECTION: Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads
78. Briefly spin the tube, and put the tube back in the magnetic rack.
SECTION: Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads
79. 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. 
SECTION: Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads
80. Remove the tube from the magnetic rack. Elute DNA target from the beads by adding 17 µL 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 2 min at room temperature. Put the tube in the magnet until the solution is clear.
SECTION: Purification of the Ligation Reaction Using SPRIselect Beads or NEBNext Sample Purification Beads
81. Without disturbing the bead pellet, transfer 15 µL of the supernatant to a clean PCR tube and proceed to PCR enrichment.
 

SECTION: PCR Enrichment of Adaptor Ligated DNA
82. [ ! ] 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.


SECTION: PCR Enrichment of Adaptor Ligated DNA
83. Set up the PCR reaction as described below based on the type of oligos (PCR primers) used.
Option A: Forward and Reverse Primers Separate:
   Component Volume Per One Library   Adaptor Ligated DNA (Step 72) 14 µl   NEBNext Ultra II Q5 Master Mix 25 µl   Universal PCR Primer/i5 Primer*,** 5 µl   Index (X) Primer/i7 Primer*,** 5 µl   Total Volume 50 µl   
 
Option B: Forward and Reverse Primers Combined:
   Component Volume Per One Library   Adaptor ligated DNA (Step 72) 15 µl   NEBNext Ultra II Q5 Master Mix 25 µl   Index (X)/Universal Primer Mix* 10 µl   Total Volume 50 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
SECTION: PCR Enrichment of Adaptor Ligated DNA
84. Mix well by gently pipetting up and down 10 times. Quickly spin the tube in a microcentrifuge.
SECTION: PCR Enrichment of Adaptor Ligated DNA
85. Place the tube on a thermocycler with the heated lid set to 105 °C and perform PCR amplification using the following PCR cycling conditions (refer to Table 82.A and Table 82.B):
Table 82.A:
   Cycle Step Temp Time Cycles   Initial Denaturation 98°C 30 seconds 1   Denaturation 98°C 10 seconds 7–16*,**   Annealing/Extension 65°C 75 seconds   Final Extension 65°C 5 minutes 1   Hold 4°C ∞     
* 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:
   Total RNA Input Recommended PCR Cycles   1,000 ng 7–8   100 ng 11–12   10 ng           
Note:  PCR cycles are recommended based on high quality Universal Human Reference Total RNA. It may require
optimization based on the sample quality to prevent PCR over-amplification.

SECTION: Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads
86. Vortex SPRIselect Beads or NEBNext Sample Purification Beads to resuspend.
SECTION: Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads
87. Add 45 µL (0.9X) of resuspended beads to the PCR reaction (~50 µL). Mix well on a vortex mixer or by pipetting up and down at least 10 times.
 



SECTION: Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads
88. Incubate for 5 min at room temperature.
SECTION: Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads
89. 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 (~5 min), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets. 

Caution: Do not discard beads.
SECTION: Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads
90. Add 200 µL of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 s, and then carefully remove and discard the supernatant.
 

SECTION: Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads
91. Repeat   once for a total of 2 washing steps.



SECTION: Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads
92. 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. 
SECTION: Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads
93. Remove the tube from the magnetic rack. Elute the DNA target from the beads by adding 23 µL 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 2 min at room temperature. Place the tube in the magnetic rack until the solution is clear. 

SECTION: Purification of the PCR Reaction using SPRIselect Beads or NEBNext Sample Purification Beads
94. Transfer 20 µL of the supernatant to a clean PCR tube, and store at –20°C. 

SECTION: Assess Library Quality on an Agilent Bioanalyzer DNA Chip
95. Run 1 µL 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. 
SECTION: Assess Library Quality on an Agilent Bioanalyzer DNA Chip
96. Check that the electropherogram shows a narrow distribution with a peak size approximately 300 bp. 
 

SECTION: Assess Library Quality on an Agilent Bioanalyzer DNA Chip
97. Figure 94: Example of RNA library size distribution on a Bioanalyzer.
 


SECTION: Isolate RNA
1. Pulverize fresh frozen tissue in liquid nitrogen with a mortar and pestle and isolate RNA using the Quiagen All Prep Kit with final elution in 40 µL Nuclease-free water. Store at -80 °C .

SECTION: Isolate RNA
2. Using Qubit, quantify RNA concentration given in   .

SECTION: Isolate RNA
3. With Bioanalyzer, check RNA quality. Should see 2 rRNA peaks. RIN value should be above 6.