TITLE: PacBio Iso-Seq Preparation for Sequel II Systems
AUTHORS: e.gustavsson

[DESCRIPTION]
The Sequel Systems generate long reads that are well-suited for characterizing fulllength transcripts produced from high-quality RNA samples. This document describes a method to construct Iso-Seq SMRTbell® libraries for sequencing on both systems allowing detection of full-length transcripts.
This protocol describes how to perform PacBio targeted Iso-Seq

[STEPS]
SECTION: cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
1. 
 

SECTION: cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
2. For each sample and Control Mouse Liver Total RNA, combine the following reagents in separate 0.5 ml reaction tubes:

 

SECTION: cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
3. Mix contents and spin the tubes briefly in a microcentrifuge.
SECTION: cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
4. Incubate the tubes at 72 °C in a hot-lid thermal cycler for 3 min, then reduce the temperature to 42 °C for 2 min.

SECTION: cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
5. Prepare a Master Mix for all reaction tubes at room temperature by combining the following reagents in the order shown:

 

SECTION: cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
6. Aliquot 5.5 µL into each reaction tube. Mix the contents of the tubes by gently pipetting and spin the tubes briefly to collect the contents at the bottom.

SECTION: cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
7. Incubate the tubes at 42 °C for 90 min.

SECTION: cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
8. Terminate the reaction by heating the tubes at 70 °C for 10 min.

SECTION: cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
9. Dilute the first-strand reaction product by adding the appropriate volume of TE buffer (10 millimolar (mM), 0.1 millimolar (mM)):

SECTION: cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
9.1. Add 40 µL if you used total RNA as the starting material.

SECTION: cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
9.2. Add 190 µL if you used more than 0.2 µg as the starting material.

SECTION: cDNA synthesis using SMARTer™ PCR cDNA Synthesis Kit First-Strand cDNA Synthesis
9.3. Add 90 µL if you used less than 0.2 µg as the starting material.

SECTION: cDNA Amplification by LD PCR
10. For each sample and control, set up an extra reaction tube to determine the optimal number of PCR cycles. In our experience, each 100 μl reaction typically yields 1–3 μg of ds cDNA after the PCR and purification steps.

  

SECTION: cDNA Amplification by LD PCR
11. For each reaction, aliquot the appropriate volume (see Table I, above) of each diluted first-strand cDNA into a labeled 0.5 ml reaction tube. If necessary, add deionized H2O to adjust the volume to 10 µL.

SECTION: cDNA Amplification by LD PCR
12. Prepare a PCR Master Mix for all reactions, plus one additional reaction. Combine the following reagents in the order shown:

 

SECTION: cDNA Amplification by LD PCR
13. Mix well by vortexing and spin the tube briefly in a microcentrifuge.
SECTION: cDNA Amplification by LD PCR
14. Aliquot 90 µL into each tube from Step 11.

SECTION: cDNA Amplification by LD PCR
15. Cap the tube. Commence thermal cycling using the following program: 

• 95 °C 1 min 
• X number of cycles (consult Table 1). 
• 95 °C 15 s 
• 95 °C 30 s 
• 95 °C 3 min

SECTION: cDNA Amplification by LD PCR
16. Subject each tube from step 9 to 15 cycles, then pause the program. Transfer 30 µL to a second reaction tube labelled “Optimization”. Store the “Experimental” tubes at 4 °C. Using the Tester PCR tube, determine the optimal number of PCR cycles:

SECTION: cDNA Amplification by LD PCR
16.1. Transfer 5 µL from the 15 cycle PCR reaction tube to a clean microcentrifuge tube (for agarose/EtBr gel analysis).

SECTION: cDNA Amplification by LD PCR
16.2. Return the Optimization tubes to the thermal cycler. Run three additional cycles (for a total of 18) with the remaining 25 µL.

SECTION: cDNA Amplification by LD PCR
16.3. Transfer 5 µL from the 18 cycle PCR reaction tube to a clean microcentrifuge tube (for agarose/EtBr gel analysis).

SECTION: cDNA Amplification by LD PCR
16.4. Run three additional cycles (for a total of 21) with the remaining 20 µL.

SECTION: cDNA Amplification by LD PCR
16.5. Transfer 5 µL from the 21 cycle PCR to a clean microcentrifuge tube (for agarose/EtBr gel analysis).

SECTION: cDNA Amplification by LD PCR
16.6. Run three additional cycles (for a total of 24) with the remaining 15 µL.

SECTION: cDNA Amplification by LD PCR
16.7. Transfer 5 µL from the 24 cycle PCR to a clean microcentrifuge tube (for agarose/EtBr gel analysis).

SECTION: cDNA Amplification by LD PCR
16.8. Run three additional cycles (for a total of 27) with the remaining 10 µL.

SECTION: cDNA Amplification by LD PCR
17. Electrophorese each 5 µL aliquot of the PCR reaction alongside 0.1 µg on a 1.2% agarose/EtBr gel in 1X TAE buffer. Determine the optimal number of cycles required for each experimental and control sample.

SECTION: cDNA Amplification by LD PCR
18. Retrieve the 15 cycle Experimental PCR tubes from 4 °C, return them to the thermal cycler, and subject them to additional cycles, if necessary, until you reach the optimal number.

SECTION: cDNA Amplification by LD PCR
19. When the cycling is completed, analyse a 5 µL alongside 0.1 µg on a 1.2% agarose/EtBr gel in 1X TAE buffer. Compare your results to Figure 1 to confirm that your reactions were successful.
  

SECTION: cDNA Amplification by LD PCR
20. Add 2 µL to each tube to terminate the reaction.

SECTION: Purification of Amplified cDNA
21. Add 1X AMPure PB beads to the amplified cDNA.
SECTION: Purification of Amplified cDNA
22. Mix by tapping the LoBind tube until the sample is homogeneous.
SECTION: Purification of Amplified cDNA
23. Incubate at 4 Room temperature for 10 min.

SECTION: Purification of Amplified cDNA
24. Place on magnetic rack until solution clears. Remove and discard supernatant.
SECTION: Purification of Amplified cDNA
25. With the tube still on magnet, add 200 µL to the tube containing beads plus DNA (1/2).

SECTION: Purification of Amplified cDNA
26. Remove and discard 70% ethanol (1/2).
SECTION: Purification of Amplified cDNA
27. With the tube still on magnet, add 200 µL to the tube containing beads plus DNA (2/2).

SECTION: Purification of Amplified cDNA
29. Let beads air dry for 1 min.

SECTION: Purification of Amplified cDNA
30. Add 27 µL and remove the tube from the magnet. Mix by tapping the tube until the sample is homogeneous. Then incubate at 4 Room temperature for 2 min.

SECTION: Purification of Amplified cDNA
31. Place back on magnet. When the solution clears, remove 25 µL into new 1.5 mL LoBind tube.

SECTION: Purification of Amplified cDNA
32. Determine concentration using Qubit device or similar quantification assay.
SECTION: Purification of Amplified cDNA
33. Run 1 µL on Agilent DNA 12000 chip according to manufacturer’s instructions.

SECTION: Purification of Amplified cDNA
34. The captured cDNA is now ready for SMRTbell library construction.
SECTION: Repair DNA Damage
35. In a LoBind microcentrifuge tube, add the following reagents:
 

SECTION: Repair DNA Damage
36. Pipette mix 10 times. It is important to mix well. Perform a quick spin to collect all liquid from the sides of the tube.
SECTION: Repair DNA Damage
37. Place in a thermocycler and run the following program: 
• 37 °C 30 min 
• Hold at 4 °C

SECTION: End Repair/A-Tailing
38. In a LoBind microcentrifuge tube, add the following reagents:

 

SECTION: End Repair/A-Tailing
39. Pipette mix 10 times. It is important to mix well. Perform a quick spin to collect all liquid from the sides of the tube.
SECTION: End Repair/A-Tailing
40. Place in a thermocycler and run the following program: 
• 20 °C 30 min 
• 65 °C 20 min 
• Hold at 4 °C

SECTION: Overhang Adapter Ligation
41. Add the following directly to reaction mix from previous step:

 

SECTION: Overhang Adapter Ligation
42. Pipette mix 10 times. It is important to mix well. Perform a quick spin to collect all liquid from the sides of the tube.
SECTION: Overhang Adapter Ligation
43. Place in a thermocycler and run the following program: 
• 20 °C 60 min 
• Hold at 4 °C

SECTION: Purification of cDNA
44. Add 1X AMPure PB beads to the amplified cDNA.
SECTION: Purification of cDNA
45. Mix by tapping the LoBind tube until the sample is homogeneous.
SECTION: Purification of cDNA
46. Incubate at 4 Room temperature for 10 min.

SECTION: Purification of cDNA
47. Place on magnetic rack until solution clears. Remove and discard supernatant.
SECTION: Purification of cDNA
48. With the tube still on magnet, add 200 µL to the tube containing beads plus DNA (1/2).

SECTION: Purification of cDNA
49. Remove and discard 70% ethanol (1/2).
SECTION: Purification of cDNA
50. With the tube still on magnet, add 200 µL to the tube containing beads plus DNA (2/2).

SECTION: Purification of cDNA
52. Remove ethanol.
SECTION: Purification of cDNA
53. Check for any remaining droplets in the tube. If droplets are present spin down down and place tube back on magnetic rack and pipette of any remaining ethanol.
SECTION: Purification of cDNA
54. Let tube air dry for 1 min.

SECTION: Purification of cDNA
55. Add 30 µL and remove the tube from the magnet. Mix by tapping the tube until the sample is homogeneous. Then incubate at 4 Room temperature for 2 min.

SECTION: Purification of cDNA
56. Place back on magnet. When the solution clears, remove 30 µL into new 1.5 mL LoBind tube.

SECTION: Purification of cDNA library
57. Perform two rounds of Ampure PB bead clean up in the "Purification of cDNA" section.
SECTION: Prepare for Sequencing
58. Follow the SMRT Link Sample Setup v8.0 (or higher) instructions for preparing the sample for sequencing on the Sequel II System.
SECTION: Purification of Amplified cDNA
28. Remove and discard 70% ethanol (2/2).
SECTION: Purification of cDNA
51. Remove and discard 70% ethanol (2/2).