TITLE: CTLR-Seq Protocol
AUTHORS: Bo Zhou, GiWon Shin, Yiling Huang, Raegan N. Wood, Hanlee P. Ji, Alexander E. Urban

[DESCRIPTION]
We developed a generally applicable method CRISPR/Cas9-targeted long read sequencing (CTLR-Seq) to resolve, haplotype-specifically, and at base-pair resolution, large, complex, and highly repetitive genomic regions that had been previously impenetrable to next-generation sequencing analysis, i.e. large segmental duplication (SegDup) regions and their associated genome rearrangements that often stretch hundreds of kilobases.  CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to haplotype-specifically isolate intact large (100-2000 kb) regions that encompass previously unresolvable genomic sequences.  These targets are then sequenced (amplification-free) with up to 250x on-target coverage using nanopore sequencing, allowing for their complete sequence assembly.

[BEFORE_START]
This protocol takes at least 5 days to complete, due to overnight incubation steps. 

[GUIDELINES]
High Molecular Weight (HMW) DNA is long and fragile. Pipetting DNA elutions should always be very slow and very gentle, using wide orifice (WO) pipette tips. 
Over-drying beads during cleanup steps will result in a decreased yield. 
Use a fresh box of pipette tips for steps involving RNA. 

Additional Notes: 

1.	Custom gRNAs are assay-specific. Design gRNAs to be specific to targets.
2.	Sage Science recommends NEB EnGen Spy Cas9 HF1 for the Cas9 nuclease, but NEB wild-type Cas9 nuclease was used in this protocol.
3.	Sage Science recommends the final total concentration of Cas9 complexes to be at 1uM in 1X HLS Enzyme Buffer.
4.	Sage Science’s PCR protocol does not call for dilution, but instead uses bCD reagent at 0.1%.

Data Analysis
 
required software:
 
minimap2 (version 2.26)
 
https://github.com/lh3/minimap2
 
seqtk 
 
https://github.com/lh3/seqtk
 
samtools (version 1.19)
 
https://github.com/samtools/
 
pod5 (version 3.10)
 
https://github.com/nanoporetech/pod5-file-format

 
flye (vesion 2.6)
 
https://github.com/fenderglass/Flye
 
dorado (version 0.5.3)
 
https://github.com/nanoporetech/dorado
 
bedtools 
 
https://github.com/arq5x/bedtools2
 

 


[STEPS]
SECTION: Sample Handling:
1. Count number of cells in sample.
SECTION: Sample Handling:
2. Wash cells 3X with phosphate-buffer



 saline (PBS). Centrifugation should be done at 100 g-200 g for 5 min-10 min, depending on cell type.

SECTION: Sample Handling:
3. Resuspend pellet in 60 µL of Sage Science M2 Buffer per million cells counted prior to washing.
SECTION: Sample Handling:
4. Store on ice.
SECTION: Qubit Quantification:
5. Gently mix the cell suspension, slowly with a WO pipette tip, to encourage solution homogeny.
SECTION: Qubit Quantification:
6. Obtain two 1.5 mL Eppendorf tubes and transfer 10 µL of cell suspension to each of the two tubes.

SECTION: Qubit Quantification:
7. Add 190 µL of Qubit Lysis Buffer to each tube, and vortex at maximum speed for 45 s.
SECTION: Qubit Quantification:
8. Briefly spin down (<1 s).
SECTION: Qubit Quantification:
9. Add 600 µL of TE buffer to each tube and vortex at maximum speed for 45 s.
SECTION: Qubit Quantification:
10. Briefly spin down (<1 s).

SECTION: Qubit Quantification:
11. Add 5 µL of lysate from each tube to a corresponding Qubit Assay tube, along with 195 µL of Qubit 1X dsDNA High Sensitivity Working Solution. 
SECTION: Qubit Quantification:
12. Briefly vortex (~3 s) to mix and spin down. 
SECTION: Qubit Quantification:
13. Incubate at Room temperature for 2 min before quantifying.

SECTION: Qubit Quantification:
14. Quantify with Qubit machine for 1x dsDNA High Sensitivity and record the concentration per mL.
SECTION: Qubit Quantification:
15. Calculate the average concentration between the two replicates.
SECTION: Qubit Quantification:
16. Multiply the average concentration per mL by 3,200 to calculate the total concentration of DNA in the sample.
SECTION: Qubit Quantification:
17. Dilute the cell suspension in M2 buffer so that 70 µL contains up to 5 µg of genomic DNA.

SECTION: Sage Science HLS CATCH
18. Prepare cassettes:

Prepare cassettes in accordance with the Sage Science HLS-CATCH Protocol.
Select the positions being used and run the “check current” protocol.
SECTION: Extraction
19. Remove the contents of the elution wells and replace with 80 µL running buffer.
SECTION: Extraction
20. Remove the contents of the sample well and replace with 70 µL of cell suspension.
SECTION: Extraction
21. Remove the contents of the reagent well and replace with 180 µL of 3% SDS HLS Lysis Reagent.
SECTION: Extraction
22. Tape the cassettes and close the lid.
SECTION: Extraction
23. Run the appropriate workflow depending on size of target of interest (e.g., workflow ‘CATCH 300-1000kb inj80V 2m sep3h.shflow.’)
SECTION: Extraction
24. After approximately one hour of run time, begin preparing the guide RNAs (can also be prepared up to 72 hours prior to start of experiment and store

 at4 °C).
SECTION: Guide RNA Preparation
25. Ensure that the tracrRNA and gRNA is diluted in duplex buffer to 100 1534 (recommended to measure RNA concentration using Qubit RNA kits to ensure correct concertation.
SECTION: Guide RNA Preparation
26. In clean RNase-free PCR tubes, prepare the following on ice: 

 

SECTION: Guide RNA Preparation
27. crRNA pools can also be used for multiple target enrichment, but adjust volume accordingly to not exceed what is recommended below.

 
 
SECTION: Guide RNA Preparation
28. Mix well and spin down.
SECTION: Guide RNA Preparation
29. Incubate the guide RNAs at 95 °C for 5 min.

SECTION: Guide RNA Preparation
30. Allow the guide RNAs to cool at Room temperature for 5 min.
SECTION: Guide RNA Preparation
31. Add the following to the respective PCR tubes: 
 
 

SECTION: Guide RNA Preparation
32. Mix well and spin down.
SECTION: Guide RNA Preparation
33. Incubate the tubes at 37 °C for 10 min.
SECTION: Guide RNA Preparation
34. Combine Tube 1 and Tube 2 as Cas9 complex mixture and leave on ice (or 4 °C for longer storage until needed).

SECTION: Guide RNA Preparation
35. When ready for treatment step below (i.e., 5-10 minutes prior to start of Treatment step below), dilute Cas9 complex mixture 4-fold with 1x Enzyme Buffer (e.g., add 240 µL of 1x Enzyme Buffer to 80 µL of Cas9 complex mixture).
SECTION: Treatment
36. After the extraction phase is complete, remove 5 mL of buffer from the (+) electrode port of each  cassette lane, where the SDS is concentrated, and replace with 5mL of fresh running buffer.
SECTION: Treatment
37. Carefully remove the tape from each cassette (slowly peel starting from the upper right corner to avoid contaminating elution wells) and remove the contents of the sample and reagent wells. 
SECTION: Treatment
38. Add 80 µL of the diluted Cas9 complex mixture to the sample wells of each cassette. 
SECTION: Treatment
39. Add 220 µL of Sage 1X enzyme buffer to the reagent wells of each cassette. 
SECTION: Treatment
40. Make sure that there is no meniscus or protrusion by adding or removing running buffer from cassettes if needed.
SECTION: Treatment
41. Close the lid and press ‘OK.’

 

SECTION: Treatment
42. Injection of Cas9 complex will start and last for approximately 2 min-4 min (depending on selected workflow. When complete, open the lid and remove the contents of the sample wells.

SECTION: Treatment
43. Add 80 µL of enzyme buffer to the sample wells. Do not tape the cassettes. 
SECTION: Treatment
44. Close the lid and press ‘OK.’
SECTION: Treatment
45. After 30 minutes, the machine will pause. Open the lid and empty the reagent wells. 
SECTION: Treatment
46. Add 180 µL of SAGE SCIENCE 1% SDS Lysis Reagent to the reagent wells.
SECTION: Treatment
47. Seal the cassettes with tape. 
SECTION: Treatment
48. Top off the running buffer such that there is no meniscus or protrusion.
SECTION: Treatment
49. Close the lid and press 'OK.'
SECTION: Collection
50. The run will complete after several hours. For optimal elution, allow the cassettes to sit undisturbed 4 min. 
SECTION: Collection
51. Open the lid and remove the tape from the cassettes.
SECTION: Collection
52. Use a WO pipette tip to remove the contents of the elution wells. Pipette very slowly by hand. 
SECTION: Collection
53. Place the contents into labeled PCR strip tubes. 
SECTION: qPCR Assay
54. Perform a 1:30 dilution by adding 1 µL of elution to 29 µL of water.  
SECTION: qPCR Assay
55. Briefly vortex the dilutions to mix. Spin down. 
SECTION: qPCR Assay
56. Prepare Master Mixes according to the following volumes: 
SECTION: qPCR Assay
57. Beta-cyclodextrin (bCD) is provided in HLS-CATCH Cassette Kit (Sage Science). 
SECTION: qPCR Assay
58.  
 
 
SECTION: qPCR Assay
59. Prepare one master mix for each probe used (using 3 probes here as example).
SECTION: qPCR Assay
60. Add the master mixes to a 96 amp FAST plate according to the following layout:
 
 

SECTION: qPCR Assay
61. Add 2 µL of dilution and 2.5 µL control DNA according to the schematic above.
SECTION: qPCR Assay
62. Mix with a pipette set to 8µL.
SECTION: qPCR Assay
63. Seal plate and spin down.
SECTION: qPCR Assay
64. Run qPCR protocol on StepOne according to the following protocol: 
SECTION: qPCR Assay
64.1. Denature 95 °Cfor 10 min.
SECTION: qPCR Assay
64.2. 50 cycles of 15 s at 95 °C and 1 min at 60 °C.
SECTION: Qubit Measurement
65. Based on the results of the qPCR, combine the elutions that have target enrichment in an Eppendorf LoBind 1.5 mL tube. 
SECTION: Qubit Measurement
66. Quantify target elution using Qubit 1X dsDNA High Sensitivity kit. Combine elution modules with target enrichment if appropriate.
SECTION: Hi Bead Cleanup
67. Bring Binding Buffer and Hi-Bead suspension to Room temperature.
SECTION: Hi Bead Cleanup
68. Add an equivalent volume of Binding Buffer to the combined elution. Allow the Binding Buffer to gently drip into the elution.  
SECTION: Hi Bead Cleanup
69. Gently incorporate the buffer into the elution by rocking back and forth.  Avoid flicking or tapping.  
SECTION: Hi Bead Cleanup
70. Place the tube in a rotator at a 45° - 90° angle to ensure gentle mixing. Mix on the rotator for 5 min.
SECTION: Hi Bead Cleanup
71. Vortex the Hi-Bead suspension. 
SECTION: Hi Bead Cleanup
72. Add the Hi-Bead Suspension in a 0.6:1 ratio of bead volume to elution volume. Add the beads to the side of the tube and allow it to gently drip into the elution.
SECTION: Hi Bead Cleanup
73. Gently rock the tube back and forth 5 times to incorporate the beads.
SECTION: Hi Bead Cleanup
74. Place the tube in the rotator at a 45° - 90° angle for 20 min.
SECTION: Hi Bead Cleanup
75. Briefly spin (<1 s), and place on a magnet.
SECTION: Hi Bead Cleanup
76. When the solution is clear, discard the supernatant.
SECTION: Hi Bead Cleanup
77. Add 80% ethanol to the tube in a 3:1 ratio of ethanol volume to elution volume. Let it sit for 3 min.

SECTION: Hi Bead Cleanup
78. Remove the supernatant.
SECTION: Hi Bead Cleanup
79. Repeat the wash step for a total of 2 washes.
SECTION: Hi Bead Cleanup
80. Spin the tube and re-magnetize to remove residual ethanol. Do not allow the beads to dry out.
SECTION: Hi Bead Cleanup
81. Add 50 µL 10mM Tris buffer and gently rock back and forth to resuspend the beads. 
SECTION: Hi Bead Cleanup
82. Incubate at 55 °C for 10 min.
SECTION: Hi Bead Cleanup
83. Decrease the heat to 37 °C and shake at   for 15 min.
SECTION: Hi Bead Cleanup
84. Gently resuspend the beads using a magnet and gentle rocking.
SECTION: Hi Bead Cleanup
85. Shake at  .
SECTION: Hi Bead Cleanup
86. Place the tube at 4 °C 15 min.
SECTION: Hi Bead Cleanup
87. Magnetize the beads to disturb the bead pellet and gently rock the tube back and forth to resuspend the beads.
SECTION: Hi Bead Cleanup
88. Magnetize and transfer the supernatant (50 µL) to a pcr tube by pipetting very slowly with a WO pipette tip.
SECTION: Hi Bead Cleanup
89. Quantify elution using Qubit 1X dsDNA High Sensitivity kit.
SECTION: ER&amp;AT
90. Add 7 µL of KAPA ERAT buffer to the 50µL of eluted DNA from the beads.
SECTION: ER&amp;AT
91. Add 3 µLERAT enzyme to the DNA, such that the total volume now is 60µL.
SECTION: ER&amp;AT
92. Mix well by pipetting very slowly with a WO pipette tip.
SECTION: ER&amp;AT
93. Incubate at 20 °C for 30 min and then at 65 °C for 30 min.
SECTION: Ligation
94. Mix the Ligation buffer (LNB) because it is viscous.

SECTION: Ligation
95. Add 25 µL of LNB to the 60 µL ERAT product.

SECTION: Ligation
96. Add 10 µL of KAPA Ligase.

SECTION: Ligation
97. Add 5 µL of Ligation Adapter (LA).

SECTION: Ligation
98. Mix well by pipetting very slowly with a WO pipette tip.
SECTION: Ligation
99. Incubate at 20 °C for 300 min and then at 4 °C 300 min.
SECTION: AMPure XP Bead Cleanup
100. Transfer the ligation product from the PCR strip tube to a 2.0 mL round bottom LoBind tube. The total volume is 100µL.
SECTION: AMPure XP Bead Cleanup
101. Vortex AMPure XP Beads (AXP) and add 50 µL AXP beads to the ligation product. 
SECTION: AMPure XP Bead Cleanup
102. Gently rock the tube back and forth to incorporate. 
SECTION: AMPure XP Bead Cleanup
103. Mix on the rotator at a 45° – 90° degree angle for 20 min. 
SECTION: AMPure XP Bead Cleanup
104. Spin down and magnetize.
SECTION: AMPure XP Bead Cleanup
105. Once the solution is clear, remove the supernatant.
SECTION: AMPure XP Bead Cleanup
106. Add 250 µL of Long Fragment Buffer (LFB) and gently rock the tube back and forth to resuspend the beads.
SECTION: AMPure XP Bead Cleanup
107. Spin down and return the tube to the magnet. 
SECTION: AMPure XP Bead Cleanup
108. Once the solution is clear, discard the supernatant.
SECTION: AMPure XP Bead Cleanup
109. Add another 250 µL of LFB and repeat the previous wash step for a total of 2 washes. 
SECTION: AMPure XP Bead Cleanup
110. Discard the supernatant and resuspend the pellet in 33 µL of Elution Buffer (EB).
SECTION: AMPure XP Bead Cleanup
111. Place the tube on the rotator horizontally for 5 min. 
SECTION: AMPure XP Bead Cleanup
112. Spin down and incubate at 37 °C for 15 min with   shaking.
SECTION: AMPure XP Bead Cleanup
113. Gently resuspend the beads using a magnet and gentle rocking.
SECTION: AMPure XP Bead Cleanup
114. Shake at  . 
SECTION: AMPure XP Bead Cleanup
115. Place the tube at 4 °C 15 min.
SECTION: AMPure XP Bead Cleanup
116. Magnetize the beads for at least 1 min.
SECTION: AMPure XP Bead Cleanup
117. Transfer 33 µL of supernatant to a new 1.5 mL LoBind tube.
SECTION: Qubit Measurement
118. Quantify target elution using Qubit 1X dsDNA High Sensitivity kit (optional).
SECTION: Priming and Loading the Flow Cell
119. Allow an Oxford Nanopore R10 Flow Cell to reach Room temperature. 
SECTION: Priming and Loading the Flow Cell
120. Insert the flow cell into the ONT promethION Sequencing Machine. 
SECTION: Priming and Loading the Flow Cell
121. Select the flow cell position and start the flow cell check.
SECTION: Priming and Loading the Flow Cell
122. Thaw, vortex, and spin down the Sequencing Buffer (SB), Library Beads (LIB) and Flow Cell Tether (FCT). 
SECTION: Priming and Loading the Flow Cell
123. Prepare the Priming Mix according to the following table: 
 
 
 
SECTION: Priming and Loading the Flow Cell
124. Record the number of available pores from the flow cell check. 
SECTION: Priming and Loading the Flow Cell
125. Open the flow cell port and turn a P1000 to 200µL.
SECTION: Priming and Loading the Flow Cell
126. Slowly rotate up the volume from 200 µL to 230 µL so that a small amount of liquid enters the tip. Discard the extracted liquid.
SECTION: Priming and Loading the Flow Cell
127. Add 550 µL of priming mix, and incubate for 5 min. 
SECTION: Priming and Loading the Flow Cell
128. Mix the Library Beads (LIB) well by pipette. 
SECTION: Priming and Loading the Flow Cell
129. Prepare the Library according to the following table: 
 
 
 
SECTION: Priming and Loading the Flow Cell
130. Rotate up another small volume from the flow cell port and discard the liquid. 
SECTION: Priming and Loading the Flow Cell
131. Add another 550 µL of priming mix and incubate for 5 min. 
SECTION: Priming and Loading the Flow Cell
132. Enter the run parameters such that the following are selected: 
DNA
PCR-free
LSK114 kit
Minimum read length 1000
High accuracy base calling
99 hour run time
SECTION: Priming and Loading the Flow Cell
133. Rotate up another small volume from the flow cell port and discard the liquid. 
SECTION: Priming and Loading the Flow Cell
134. Load the 200µL Library into the flow cell port. 
SECTION: Priming and Loading the Flow Cell
135. Allow the Library to incubate in the flow cell for 60 min. 
SECTION: Priming and Loading the Flow Cell
136. Begin sequencing.