TITLE: NGS library preparation using Ovation RNA-Seq System V2 (M01206 v9) and Ovation Ultralow System V2 (M01437 v2) for animal tissue samples
AUTHORS: Ine Boonen, Magda Bletsa, Yiqiao Li, Philippe Lemey

[DESCRIPTION]
This protocol is used for successful NGS library preparation from total RNA of animal tissue samples. 
With this method we obtained full-length paramyxovirus genomes using the Illumina sequencing platform. It works most efficiently on RNA viruses possessing a polyA tail, but it can also be used for sequencing any other RNA viral sequences originating from tissue samples. 

[BEFORE_START]
Check RNA sample quality prior to library preparation. RNA sample quality may vary between sample preparations. 
Ensure pipettes are properly calibrated as library preparations are highly sensitive to pipetting error.

[GUIDELINES]
Thaw components used in each step and immediately place them on ice. Always keep thawed reagents and reaction tubes on ice unless otherwise instructed. Do not thaw all reagents at once. 
After thawing and mixing buffer mixes, if any precipitate is observed, re-dissolve the precipitate completely prior to use. You may gently warm the buffer mix for 2 minutes at room temperature followed by brief vortexing. 
Keep enzyme mixes on ice after briefly spinning to collect the contents. Do not vortex enzyme mixes nor warm any enzyme or primer mixes.
When instructed to mix via pipetting, gently aspirate and dispense a volume that is at least half of the total volume of the reaction mix.
Use only fresh ethanol stocks to prepare the dilutions of ethanol washes throughout the purification protocols.
Return all reagents to their appropriate storage conditions promptly after use unless otherwise instructed.
Total RNA input must be between 500 pg and 100 ng.
RNA samples must be free of contaminating proteins and other cellular material, organic solvents (including phenol and ethanol) and salts used in many RNA isolation methods. If a method such as Trizol is used, we recommend using a column purification after isolation.

[STEPS]
SECTION: B. First Strand cDNA Synthesis
6. Remove the First Strand Primer Mix (blue: A1) and First Strand Buffer mix (Blue: A2) from the freezer. Let it thaw at Room temperature. Mix by vortexing, spin down and place on ice.

SECTION: A. Sample Preparation for Ovation RNA-Seq system V2
1. Remove Nuclease-free water (Green: D1) from the freezer and let it thaw at Room temperature .

SECTION: F. Purification of SPIA cDNA
44. For purification the   was used following the manufacturer’s guidelines. This kit was chosen due to its low elution.
IMPORTANT!: 
- Buffer ERC is considered hazardous according to QIAGEN, and an SDS should be consulted.
- Add the appropriate amount of 100% ethanol to Buffer PE before use (see bottle label for volume). Failure to do so will result in low amplification yields.
- All centrifugation steps are carried out at maximum speed in a conventional tabletop microcentrifuge at Room temperature.

SECTION: E. SPIA Amplification
34. Remove the SPIA Primer Mix (Red: C1) and the SPIA Buffer Mix (Red: C2) from the freezer. Let it thaw at Room temperature. Mix by vortexing, spin down and place on ice. 

SECTION: D. cDNA Purification
22. Ensure the Agencourt beads have completely reached Room temperature before proceeding.

SECTION: C. Second strand cDNA Synthesis
15. Remove Agencourt beads from the fridge and place on the bench top to reach Room temperature for use in the next step. (At least 30 min before section D)

SECTION: G. SPIA cDNA Yield and Purity
60. Mix the purified SPIA cDNA sample by brief vortexing. Spin down contents.
SECTION: A. Sample Preparation for Ovation RNA-Seq system V2
3. Aliquot each total RNA input sample (500 pg-100 ng) into a 0.2 ml strip tube or a 96-well plate. Input volume can not exceed 5 µL.

SECTION: A. Sample Preparation for Ovation RNA-Seq system V2
4. Dilute the RNA with D1 to a final volume of 5 µL.

SECTION: A. Sample Preparation for Ovation RNA-Seq system V2
5. Note: Keep D1 at Room temperature for use in next sections

SECTION: B. First Strand cDNA Synthesis
7. Remove First Strand Enzyme Mix (blue: A3) from the freezer, spin down and place on ice.
SECTION: B. First Strand cDNA Synthesis
8. Add 2 µL of A1 to 5 µL of each input RNA for a total of 7 µL. Mix by pipetting 5 times, spin down and place on ice.

SECTION: B. First Strand cDNA Synthesis
9. Place the tubes in a preheated thermal cycler programmed to run the Primer annealing program: 
 


SECTION: B. First Strand cDNA Synthesis
10. Remove the tubes from the thermal cycler and place on ice.
SECTION: B. First Strand cDNA Synthesis
11. Once the Primer annealing (step 8) is complete, prepare a master mix of A2 and A3 as follows:  
 
Mix well by pipetting 5 times, spin down and place on ice.
SECTION: B. First Strand cDNA Synthesis
12. Add 3 µL of First Strand Master Mix to each sample tube for a total of 10 µL. Mix by pipetting 5 times, spin and place on ice.

SECTION: B. First Strand cDNA Synthesis
13. Place the tubes in a preheated thermal cycler programmed to run the First Strand synthesis program: 
 


SECTION: B. First Strand cDNA Synthesis
14. Remove the tubes from the thermal cycler, spin to collect all droplets from the wall of the tubes and place them on ice.
SECTION: C. Second strand cDNA Synthesis
16. Remove the Second Strand Buffer Mix (Yellow: B1) from the freezer. Let it thaw at Room temperature. Mix by vortexing, spin down and place on ice.

SECTION: C. Second strand cDNA Synthesis
17. Remove Second Strand Enzyme Mix (Yellow: B2) from the freezer, spin down and place on ice.
SECTION: C. Second strand cDNA Synthesis
18. Prepare a master mix of B1 and B2 as follows: 
 
Mix well by pipetting 5 times, spin down and place on ice.
SECTION: C. Second strand cDNA Synthesis
19. Add 10 µL of Second Strand Master Mix to each sample tube for a total of 20 µL Mix well by pipetting 5 times, spin down and place on ice.

SECTION: C. Second strand cDNA Synthesis
20. Place the tubes in a preheated thermal cycler programmed to run the Second Strand synthesis program: 
 

SECTION: C. Second strand cDNA Synthesis
21. Remove the tubes from the thermal cycler, spin to collect all droplets from the wall of the tubes and place them in a rack on the bench top.
SECTION: D. cDNA Purification
23. Prepare a 70% Ethanol wash solution

NOTE:It is critical this solution to be prepared fresh on the same day of the experiment from a recently opened stock container.
Measure both water and ethanol components carefully prior to mixing. Failure to do so can result in a higher than anticipated aqueous content, which may reduce amplification yield.

SECTION: D. cDNA Purification
24. Resuspend the beads by vortexing. Ensure that the beads are fully resuspended before adding them to the sample. After resuspending DO NOT spin the beads
SECTION: D. cDNA Purification
25. At Room temperature, add 32 µL (1.6 volumes) of Agencourt beads to each reaction and mix by gently pipetting up and down 10 times.

SECTION: D. cDNA Purification
26. Incubate at Room temperature for 10 min

SECTION: D. cDNA Purification
27. Transfer tubes to the magnet and let them stand for 5 min until the solution of the beads seems completely clear.

SECTION: D. cDNA Purification
28. Keeping the tubes on the magnet, add 200 µL of freshly prepared 70% ethanol and allow to stand for 30 s.
NOTE: The beads should not disperse; instead, they will stay on the walls of the tubes. Significant loss of beads at this stage will impact cDNA yields, so ensure beads are not removed with the binding buffer or the washes.

SECTION: D. cDNA Purification
29. Remove the 70% ethanol wash using a pipette.
SECTION: D. cDNA Purification
30. Repeat the previous washing steps two more times.
SECTION: D. cDNA Purification
31. With the final wash, it is critical to remove as much of the ethanol as possible. Use at least 2 pipetting steps and allow excess ethanol to collect at the bottom of the tubes after removing most of the ethanol in the first pipetting step. Try to remove last bits of ethanol with a smaller pipette. 
SECTION: D. cDNA Purification
32. Air-dry the bead on the magnet for 15 - 20 min. Inspect each tube carefully to ensure that all the ethanol has evaporated.
IMPORTANT!: It is critical that all residual ethanol be removed prior to continuing with SPIA amplification.
SECTION: D. cDNA Purification
33. Continue immediately to the SPIA Amplification protocol with the cDNA still bound to the dry beads.
SECTION: E. SPIA Amplification
35. Remove SPIA Enzymer Mix (Red: C3) from the freezer, mix the contents by inverting gently 5 times without introducing bubbles. Spin down and place on ice.
SECTION: E. SPIA Amplification
36. Prepare the SPIA master mix by sequentially combining C2, C1 and C3 as follows:
 
NOTE: Make sure to add C3 last.
SECTION: E. SPIA Amplification
37. Add 40 µL of the SPIA Master Mix to each tube containing the double-stranded cDNA bound to the dried beads. Use a pipette set to 30 µL and mix thoroughly by pipetting at least 8-10 times. Attempt to get the majority of the beads in suspension and remove most of the beads from the tube walls.
NOTE: The beads may not form a perfectly uniform suspension, but this will not affect the reaction. The addition of SPIA master mix will elute the cDNA from the beads.

SECTION: E. SPIA Amplification
38. Place the tubes in a pre-warmed thermal cycler programmed to run the SPIA amplification program: 

 

SECTION: E. SPIA Amplification
39. Remove the tubes from thermal cycler, spin to collect condensation and place on ice.
SECTION: E. SPIA Amplification
40. IMPORTANT!: At this point the tubes should be removed from the pre-amplification workspace. Carry out all remaining steps in a post-amplification workspace using dedicated post-amplification consumables and equipment. Take care to avoid the introduction of previously amplified cDNA into your pre-amplification workspace.
SECTION: E. SPIA Amplification
41. Transfer tubes to the magnet and let them stand for 5 min to completely clear the solution of beads.

SECTION: E. SPIA Amplification
42. Carefully transfer 40 µL of the cleared supernatant containg the SPIA cDNA to a fresh tube.

SECTION: E. SPIA Amplification
43. Continue immediatly with the Purification of SPIA cDNA protocol or store the reaction products at -20 °C.

SECTION: F. Purification of SPIA cDNA
56. Let the column stand for 1 min at Room temperature.

SECTION: F. Purification of SPIA cDNA
45. Add 300 µL of Buffer ERC from the QIAGEN kit into a clean, labeled 1.5 ml microcentrifuge tube.
SECTION: F. Purification of SPIA cDNA
46. Transfer the entire volume (40 µL) of the SPIA reaction to the 1.5 ml tube containing buffer ERC.
SECTION: F. Purification of SPIA cDNA
47. Vortex for 5s and then spin briefly.
SECTION: F. Purification of SPIA cDNA
48. Label a MinElute spin column and place it into a collection tube.
SECTION: F. Purification of SPIA cDNA
49. Load the sample/buffer mixture onto the column and centrifuge 1 min  at maximum speed.
SECTION: F. Purification of SPIA cDNA
50. Place the column in a new collection tube and discard the old collection tube with the flow-through.
SECTION: F. Purification of SPIA cDNA
51. Add 750 µl of Buffer PE to the column and centrifuge for 1 min  at maximum speed.
SECTION: F. Purification of SPIA cDNA
52. Place the column in a new collection tube and discard the old collection tube with the flow-through.
SECTION: F. Purification of SPIA cDNA
53. Centrifuge the column for an additional 2 min at maximum speed to remove all residual buffer PE.
SECTION: F. Purification of SPIA cDNA
54. Discard flow-through along with collection tube. Blot the column tip onto clean absorbent paper to remove any residual wash buffer from the tip of the column.
SECTION: F. Purification of SPIA cDNA
55. Place the column into a clean labeled 1.5 ml microcentrifuge tube. Add 22 µL of Room temperature 1x TE or Buffer EB to the center of each column.

SECTION: F. Purification of SPIA cDNA
57. Centrifuge for 1 min at maximum speed.

SECTION: F. Purification of SPIA cDNA
58. Measure the volume recovered. There should be approximately 20 µl of purified SPIA cDNA. Then discard the column.
SECTION: F. Purification of SPIA cDNA
59. Continue with the SPIA cDNA yield and Purity Protocol or store the purified SPIA cDNA at -20 °C 

SECTION: G. SPIA cDNA Yield and Purity
61. Measure the absorbance at 230, 260, 280 and 320 nm on a spectophotometer. You may need to make a 1:20 dilution of the cDNA in water prior to measuring the absorbance.
SECTION: G. SPIA cDNA Yield and Purity
63. Yield: 
Assume 1 A260 unit = 50 μg/mL.
To calculate: (A260–A320 of diluted sample) X (dilution factor) X 50 (concentration in μg/mL of a 1 A260 unit solution) X 0.02 (final volume in mL) = total yield in micrograms
NOTE: If there is not available a spectrophotometer, you can use any other type of quantitation system to quantify the SPIA amplification products (i.e. Qubit, QuantiFluor). 
SECTION: G. SPIA cDNA Yield and Purity
62. Purity: 
Subtract the A320 value from the A230, A260 and A280 values. The adjusted
(A260–A320) / (A280–A320) ratio should be greater than 1.8. The adjusted (A260–A320)/(A230–A320) ratio should be greater than 2.
SECTION: G. SPIA cDNA Yield and Purity
64. The purified cDNA may be stored at -20 °C

SECTION: OVATION ULTRALOW SYSTEM V2
65. The following kit was used to make libraries for sequencing from the previously prepared cDNA.  
  
IMPORTANT!: Make sure to use reagents from the right kit. The reagents in the ovation RNA-seq system V2 product can be similar to reagents in this kit, however, unless the component part numbers are identical, these reagents do not have exactly the same composition and therefore, are not interchangeable. For example A1 from one kit with A1 from another kit.
NOTE: Remove Agencourt beads from fridge and Nuclease-free Water (green: D1) from freezer and place them on bench top. 
SECTION: OVATION RNA-SEQ SYSTEM V2
2. The following kit was used to prepare cDNA from RNA prior to the library preparation step: 
Ovation RNA-SEQ System V2 (M01206 v9)
SECTION: I. End Repair
67. Remove the End Repair Buffer Mix (Blue:ER1). Let it thaw at Room temperature. Mix by vortexing, spin down and place on ice. 

SECTION: I. End Repair
68. Remove End Repair Enzyme Mix (Blue: ER2) and End Repair Enhancer (blue: ER3) from the freezer. Spin down and place on ice.
SECTION: I. End Repair
70. Prepare the End repair master mix by combining ER1, ER2 and ER3 as follows:

 

SECTION: I. End Repair
69. Obtain 10 µL of the cDNA sample (10 pg – 100 ng) that was previously stored at -20 °C. Alternatively, place  10 pg – 100 ng of DNA in 10 µL of low-EDTA TE buffer or Nuclease-free Water in a PCR tube. 
SECTION: I. End Repair
71. Add 5 µL of the End Repair Master Mix to each sample tube and mix by pipetting. Spin the tubes down and place on ice.

SECTION: I. End Repair
72. Place the tubes in a preheated thermal cycler programmed to run the End Repair amplification program: 
 


SECTION: I. End Repair
73. Remove the tubes from the thermal cycler, spin to collect all droplets from the wall of the tubes and place them on ice. Continue immediatly with the Ligation Protocol.
SECTION: J. Ligation
74. Remove the Ligation Buffer Mix (Yellow: L1) and Ligation Adaptor Mix (Yellow:L2) from the freezer. Let it thaw on ice. Mix by vortexing, spin down and place on ice.

SECTION: J. Ligation
75. Remove Ligation Enzyme Mix (Yellow:L3) from the freezer. Spin down and place on ice.
SECTION: J. Ligation
76. Add L2 to each sample as follows:
- If using adaptors from tubes (0344NB-08), add 6 µL of the appropriate L2 Ligation Adaptor Mix to each sample. Mix thoroughly by pipetting.
- If using an adaptor plate (0344-32, 0344NB-32 or 0344NB-A01), add the entire 15 µL of sample to the appropriate adaptor well, mix well by pipetting, then transfer the entire sample to a PCR tube.
NOTE: All samples on a sequencing run should have unique ligation adaptors.

SECTION: J. Ligation
77. Prepare the Ligation master mix by combining D1, L1 and L3 as follows: 
 
NOTE: L1 is very viscous. Please be sure to pipette this reagent slowly.
SECTION: J. Ligation
78. Add 9 µL of the Ligation Master Mix to each sample tube and mix by pipetting. Spin the tubes down and place on ice. Proceed immediatly with the incubation.

SECTION: J. Ligation
79. Place the tubes in a preheated thermal cycler programmed to run the End Repair amplification program: 

 

SECTION: J. Ligation
80. Remove the tubes from the thermal cycler, spin to collect all droplets from the wall of the tubes and place on ice. Continue immediately with the Ligation Purification Protocol.
SECTION: K. Ligation Purification
83. Add 70 µL of Room temperature D1 to each ligation reaction.

SECTION: K. Ligation Purification
84. At room temperature, add 80 µL (0.8 volumes) of the bead suspension to each tube and mix by pipetting 10 times.

SECTION: K. Ligation Purification
85. Incubate at Room temperature for 10 min.

SECTION: K. Ligation Purification
86. Transfer the PCR tubes containing the bead-sample mixture to the magnet and let them stand for5 min to completely clear the solution of beads.

SECTION: K. Ligation Purification
87. Carefully remove 160 µL of the binding buffer and discard it. Leaving some of the volume behind minimizes bead loss at this step.

SECTION: K. Ligation Purification
88. With the tubes still on the magnet, add 200 µL of freshly prepared 70% ethanol and allow to stand for 30 s.

SECTION: K. Ligation Purification
89. Remove the 70% ethanol wash using a pipette.
SECTION: K. Ligation Purification
90. Repeat the 70% ethanol wash one more time, for a total of two washes.
SECTION: K. Ligation Purification
91. With the final wash, it is critical to remove as much of the ethanol as possible. Use at least 2 pipetting steps and allow excess ethanol to collect at the bottom of the tubes after removing most of the ethanol in the first pipetting step. Try to remove last bits of ethanol with a smaller pipette. 
SECTION: K. Ligation Purification
92. Air dry the beads on the magnet for 10 min. Inspect each tube carefully to ensure that all the ethanol has evaporated.

SECTION: K. Ligation Purification
93. Add 40 µL Room temperature 1X low-EDTA TE buffer or Nuclease-free Water (green: D1) to the dried beads. Mix thoroughly to ensure all the beads are resuspended and let them stand on the bench top for 3 min
SECTION: K. Ligation Purification
95. Carefully remove 35 µL of the eluate, ensuring as few beads as possible are carried over, then transfer to a fresh set of PCR tubes and place on ice.

SECTION: K. Ligation Purification
96. Set aside the Agencourt beads and 70% ethanol at room temperature for use in the Amplified Library Purification protocol. Also set aside the Nuclease-free water (green:D1) for use throughout the protocol.
SECTION: K. Ligation Purification
97. Continue immediately to the Library Amplification Protocol.
SECTION: K. Ligation Purification
94. Transfer the tubes to the magnet and let them stand for 3 min to completely clear the solution of beads.

SECTION: L. Library Amplification
98. Remove the Amplification Buffer Mix (Red: P1), Amplification Primer Mix (Red: P2) from the freezer. Let it thaw at Room temperature. Mix by vortexing, spin down and place on ice.

SECTION: L. Library Amplification
99. Remove Amplification Enzyme Mix (Red: P3) from the freezer. Spin down and place on ice.
SECTION: L. Library Amplification
100. Prepare the Ligation master mix by sequentially combining P1 and P2. Add P3 last, as follows, and mix well, taking care to avoid bubbles. Then spin down and place on ice.

 

SECTION: L. Library Amplification
101. on ice Add 15 µL of the Amplification Master Mix to each sample tube and mix by pipetting. Spin the tubes down and place on ice. 

SECTION: L. Library Amplification
102. Place the tubes in a preheated thermal cycler programmed to run the End Repair amplification program: 

 
IMPORTANT!: The number of cycles used for PCR amplification depends on the starting amount of genomic DNA. Please refer to the table below for a general guide to choosing the appropriate number of cycles for the PCR amplification reaction. Alternatively, real-time PCR can be used to determine the appropriate number of PCR cycles.

 

SECTION: L. Library Amplification
103. Remove the tubes from the thermal cycler, spin to collect all droplets from the wall of the tubes and place on ice. Continue immediately with the Amplified Library Purification Protocol.
SECTION: M. Amplified Library Purification
104. Take Agencourt beads and 70% ethanol that we used earlier. Ensure they are atRoom temperaturebefore proceeding.

SECTION: M. Amplified Library Purification
105. Make sure beads are resuspended by inverting and tapping the tube.  After resuspending, do not spin the beads.
SECTION: M. Amplified Library Purification
106. At room temperature, add 50 µL (1 volume) of the bead suspension to each tube and mix by pipetting 10 times.

SECTION: M. Amplified Library Purification
107. Incubate at Room temperature for 10 min.

SECTION: M. Amplified Library Purification
108. Transfer the PCR tubes containing the bead-sample mixture to the magnet and let them stand for5 min to completely clear the solution of beads.

SECTION: M. Amplified Library Purification
109. Carefully remove 85 µL of the binding buffer and discard it. Leaving some of the volume behind minimizes bead loss at this step.

SECTION: M. Amplified Library Purification
110. With the tubes still on the magnet, add 200 µL of freshly prepared 70% ethanol and allow to stand for 30 s.

SECTION: M. Amplified Library Purification
111. Remove the 70% ethanol wash using a pipette.
SECTION: M. Amplified Library Purification
112. Repeat the 70% ethanol wash one more time, for a total of two washes.
SECTION: M. Amplified Library Purification
113. With the final wash, it is critical to remove as much of the ethanol as possible. Use at least 2 pipetting steps and allow excess ethanol to collect at the bottom of the tubes after removing most of the ethanol in the first pipetting step. Try to remove last bits of ethanol with a smaller pipette. 
SECTION: M. Amplified Library Purification
114. Air dry the beads on the magnet for 10 min. Inspect each tube carefully to ensure that all the ethanol has evaporated.

SECTION: M. Amplified Library Purification
115. Add 33 µL Room temperature 1X low-EDTA TE buffer or Nuclease-free Water (green: D1) to the dried beads. Mix thoroughly to ensure all the beads are resuspended.
SECTION: M. Amplified Library Purification
116. Transfer the tubes to the magnet and let them stand for 2 min to completely clear the solution of beads.

SECTION: M. Amplified Library Purification
117. Carefully remove 30 µL of the eluate, ensuring as few beads as possible are carried over, then transfer to a fresh set of PCR tubes and place on ice.

SECTION: M. Amplified Library Purification
118. Library can now be stored at -20 °C 

SECTION: H. DNA Fragmentation
66. No fragmentation step was performed, since the input cDNA fragment sizes were relatively short. Therefore, we also skipped the DNA purification step, which is normally performed after the DNA fragmentation, and directly proceeded to the end repair step.  
SECTION: K. Ligation Purification
81. Prepare a 70% ethanol wash solution. It is critical this solution to be prepared fresh on the same day of the experiment from a recently opened stock container. Measure both the ethanol and the water components carefully prior to mixing. Failure to do so can result in a higher than anticipated aqueous content, which may reduce yield.
1.5 mL wash solution per sample is enough for all the bead purification steps. 
SECTION: K. Ligation Purification
82. Ensure the Agencourt beads and Nuclease-free Water (D1) have completely reached Room temperaturebefore proceeding. Make sure beads are resuspended by inverting and tapping the tube.  After resuspending, do not spin the beads.