TITLE: snASE - MAS-Seq protocol
AUTHORS: Asa Shin, Aziz Al'Khafaji

[DESCRIPTION]
Modified version of PacBio's MAS-seq protocol for snASE samples. 

[STEPS]
SECTION: Quality Control
1. 1 Bring the Qubit 1X dsDNA HS working solution and standards to room temperature.
SECTION: Quality Control
2. 2 Pulse vortex or pipette mix each sample to homogenize the DNA in solution.
SECTION: Quality Control
3. 3 Quick spin each sample to collect liquid.
SECTION: Quality Control
4. 4 Take a 1 μL aliquot from each sample.
SECTION: Quality Control
5. 5 Measure DNA concentration with a Qubit fluorometer using the 1X dsDNA HS kit.
SECTION: Quality Control
6. 6 Dilute each sample to 1.0-1.5 ng/μL in elution buffer or water, based on the Qubit reading.
SECTION: Quality Control
7. 7 MeasureDNA size distribution with a Bioanalyzer system using the High Sensitivity DNA Kit.
SECTION: Quality Control
8. 8 Proceed to the next step of the protocol if sample quality is acceptable.
SECTION: TSO PCR
9. Prepare PCR mix per sample: 
 

SECTION: TSO PCR
10.  Pipette-mix RM1.
SECTION: TSO PCR
11.  Quick spin RM1 in a microcentrifuge to collect liquid.
SECTION: TSO PCR
12.  Select the TSO PCR program based on cDNA input.
SECTION: TSO PCR
13.  Cleanup with 1.5X SMRTbell cleanup beads
SECTION: TSO PCR
14. Add 1.5X v/v (75 μL) of resuspended, room-temperature SMRTbell cleanup beads to each tube of amplified cDNA.
SECTION: TSO PCR
15.  Pipette mix the beads until evenly distributed.
SECTION: TSO PCR
16.  Quick spin the tube strip in a microcentrifuge to collect liquid.
SECTION: TSO PCR
17. Leave at room temperature for 10 minutes to allow DNA to bind beads.
SECTION: TSO PCR
18. Place tube strip in a magnetic separation rack until beads separate fully from the solution.
SECTION: TSO PCR
19. Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant.
SECTION: TSO PCR
20. Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into each tube. After 30 seconds, pipette off the 80% ethanol and discard.
SECTION: TSO PCR
21. Repeat the previous step.
SECTION: TSO PCR
22. Remove residual 80% ethanol: • Remove tube strip from the magnetic separation rack. • Quick spin tube strip in a microcentrifuge. • Place tube strip back in a magnetic separation rack until beads separate fully from the solution. • Pipette off residual 80% ethanol and discard.
SECTION: TSO PCR
23. Remove tube strip from the magnetic rack. Immediately add 42 µL of elution buffer to each tube and resuspend the beads by pipetting 10 times or until evenly distributed.
SECTION: TSO PCR
24. Quick spin the tube strip in a microcentrifuge to collect liquid.
SECTION: TSO PCR
25. Leave at room temperature for 5 minutes to elute DNA.
SECTION: TSO PCR
26. Place tube strip in a magnetic separation rack until beads separate fully from the solution.
SECTION: TSO PCR
27. Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new tube strip. Discard old tube strip with beads.
SECTION: TSO PCR
28. Recommended: Evaluate sample concentration. Take a 1 µL aliquot from each tube. Measure DNA concentration with a Qubit fluorometer using the 1x dsDNA HS kit.
SECTION: TSO PCR
29. Proceed to the next step of the protocol if sample quantity is acceptable (at least 150 ng) and not exceeding 1 μg. If the sample quantity is higher than 1 μg, only carry forward to Step 3 with a maximum input of 1 μg
SECTION: TSO artifact removal
30. Bring MAS capture beads kit to room temperature. Resuspend the beads by vortexing.
SECTION: TSO artifact removal
31. Transfer 10 μL resuspended MAS capture beads per sample to a PCR tube. Scale up the amount of beads if processing more than 4 samples (with 10% overage). If preparing more than 40 μL of beads, use a 1.5 mL LoBind tube instead of PCR tube.
SECTION: TSO artifact removal
32. Place the tube on the magnet until the beads separate fully from the solution.
SECTION: TSO artifact removal
33. Carefully remove and discard the supernatant while the tube remains on the magnet. Avoid touching the bead pellet with the pipette tip.
SECTION: TSO artifact removal
34. Remove the tube from the magnet. 
Add 40 μL MAS bead binding buffer along the insidewall of the tube where the beads are collected and gently resuspend by pipetting using wide bore tips. 
DO NOT VORTEX.
Note: the solution may be viscous. Highly recommend using wide bore tips to avoid foaming. 
When excess bubbles are present, lower cDNA recovery is expected. 
Quick-spin the tube in a microcentrifuge if needed. 
Note: Scale up the volume of MAS capture binding buffer accordingly if preparing more than 40 µL of beads.
SECTION: TSO artifact removal
35. Place the tube on the magnet until the beads separate fully from the solution and remove the supernatant.
SECTION: TSO artifact removal
36. Resuspendthe beads in 40 μL MAS bead binding buffer by pipetting slowly using wide bore tips.
DO NOT VORTEX.
Note: the solution may be viscous. Highly recommend using wide bore tips to avoid foaming.
When excess bubbles are present, lower cDNA recovery is expected.
Note: Scale up the volume of MAS capture binding buffer accordingly, if preparing more than 40 µL of beads. 
Distribute 40 μLof resuspended MAScapture beads into the appropriate number of PCR tubes before proceeding to Step 3.8.
SECTION: TSO artifact removal
37. Add 40 μL of a solution containing the biotinylated DNA-fragments (from Step 2.18) to the resuspended beads. Mix carefully using wide bore tips to avoid foaming of the solution.
SECTION: TSO artifact removal
38. Incubate the tube at room temperature for 15 minutes on a rotatorto keep the beads in suspension. Quick-spin the tube in a microcentrifuge to collect liquid.
SECTION: TSO artifact removal
39. Place the tube on the magnet until the beads separate fully from the solution and remove the supernatant.
SECTION: TSO artifact removal
40. Resuspend the MAS capture beads/DNA-complex in 80 μL MAS bead washing buffer by pipette mixing until evenly distributed.
SECTION: TSO artifact removal
41. Place the tube on the magnet until the beads separate fully from the solution and remove the supernatant.
SECTION: TSO artifact removal
42. Remove the tube from the magnet. Resuspend the MAS capture beads/DNA-complex in 80 μL MAS bead washing buffer by pipette mixing until evenly distributed.
SECTION: TSO artifact removal
43. Place the tube on the magnet until the beads separate fully from the solution and remove the supernatant.
SECTION: TSO artifact removal
44. Remove the tube from the magnet. Resuspend the MAS capture beads/DNA complex in 80 μL nuclease free water by pipette mixing until evenly distributed.
SECTION: TSO artifact removal
45. Place the tube on the magnet until the beads separate fully from the solution and remove the supernatant.
SECTION: TSO artifact removal
46. Resuspend the capture beads/DNA-complex in 40 µL of elution buffer by pipette mixing until evenly distributed.
SECTION: TSO artifact removal
47. Add 2 µL MAS enzyme to the sample with capture beads to cleave the captured DNA products from MAS capture beads.
SECTION: TSO artifact removal
48. Pipette-mix each sample and a very quick spin in a microcentrifuge to collect liquid.
SECTION: TSO artifact removal
49. Run the TSOartifact removal program. Heated lid set at 47°C

 


SECTION: TSO artifact removal
50. Place the tube on the magnet for 1 minute and move the supernatant containing the library to a fresh tube.
SECTION: TSO artifact removal
51. Cleanup with 1.5X SMRTbell cleanup beads.
SECTION: TSO artifact removal
52. Add 1.5X v/v (63 μL) of resuspended, room-temperature SMRTbell cleanup beads to each sample.
SECTION: TSO artifact removal
53. Pipette-mix the beads until evenly distributed.
SECTION: TSO artifact removal
54. Quick-spin the tube strip in a microcentrifuge to collect liquid.
SECTION: TSO artifact removal
55.  Leave at room temperature for 10 minutes to allow DNA to bind beads.
SECTION: TSO artifact removal
56.  Place tube strip in a magnetic separation rack until the beads separate fully from the solution.
SECTION: TSO artifact removal
57.  Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant.
SECTION: TSO artifact removal
58. Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into each tube. After 30 seconds, pipette off the 80% ethanol and discard.
SECTION: TSO artifact removal
59.  Repeat the previous step.
SECTION: TSO artifact removal
60. Remove residual 80% ethanol: • Remove the tube strip from the magnetic separation rack. • Quick-spin the tube strip in a microcentrifuge. • Place the tube strip back in a magnetic separation rack until the beads separate fully from the solution. Pipette off residual 80% ethanol and discard.
SECTION: TSO artifact removal
61. Remove the tube strip from the magnetic rack. Immediately add 46 µL of elution buffer to each tube and resuspend the beads by pipetting 10 times or until evenly distributed.
SECTION: TSO artifact removal
62.  Quick-spin the tube strip in a microcentrifuge to collect liquid.
SECTION: TSO artifact removal
63. Leave at room temperature for 5 minutes to elute DNA.
SECTION: TSO artifact removal
64. Place the tube strip in a magnetic separation rack until beads separate fully from the solution.
SECTION: TSO artifact removal
65. Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new 0.5 mL LoBind tube. Discard the old tube strip with beads.
SECTION: TSO artifact removal
66. Recommended: Evaluate sample concentration. • Take a 1 µL aliquot from each tube. Measure DNA concentration with a Qubit fluorometer using the 1x dsDNA HS kit.
SECTION: TSO artifact removal
67. Proceed to the next step of the protocol if sample quantity is acceptable (maximum 50 ng). 
If cDNA amount is >50 ng, dilute the cDNA to 50 ng using elution buffer in a total volume of 45µL
SECTION: MAS PCR
68. Set up the following PCR reaction mix per sample on ice.
Reaction Mix 2 (RM2):
 

SECTION: MAS PCR
69. Quick-spin RM2 in a microcentrifuge to collect liquid.
SECTION: MAS PCR
70. Add 22.5 µL of RM2 to a new PCR tube on ice. Repeat this step to prepare a total of 16 tubes per sample (each containing 22.5 µL of RM2)
SECTION: MAS PCR
71. Add 2.5 µL of MAS primers premix A-PQ into each of 16 PCR tubes on ice, with each tube getting only one primer. Tube 1= Primer mix A, Tube 2=Primer mix B,... Tube 16= Primer mix PQ. DO NOT ADD MORE THAN ONE PRIMER PREMIX TO A TUBE
SECTION: MAS PCR
72. Pipette-mix each sample. The total volume of each tube should be 25.0 µL.
SECTION: MAS PCR
73. Quick-spin the strip tubes in a microcentrifuge to collect liquid.
SECTION: MAS PCR
74. Run the MAS PCR program. Reactions can be held overnight in the cycler.
Note: if the total sample quantity is less than 50 ng, follow the table below for cycle number.

 


SECTION: MAS PCR
75. Clean up with 1.5X SMRTbell cleanup beads.
SECTION: MAS PCR
76. Pool entire volume of all 16 reactions into a single 1.5 mL LoBind tube.
SECTION: MAS PCR
77. Add 1.5X v/v (600 µL) ofresuspended, room-temperature SMRTbell cleanup beads to the PCR pool.
SECTION: MAS PCR
78.  Pipette-mix the beads until evenly distributed.
SECTION: MAS PCR
79. Quick-spin the tube strip in a microcentrifuge to collect all liquid from the sides of the tubes.
SECTION: MAS PCR
80. Leave at room temperature for 10 minutes to allow DNA to bind beads.
SECTION: MAS PCR
81. Place 1.5mL LoBind tube in a magnetic separation rack until beads separate fully from the solution.
SECTION: MAS PCR
82. Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant.
SECTION: MAS PCR
83. Slowly dispense 1 mL, or enough to cover the beads, of freshly prepared 80% ethanol into each tube. After 30 seconds, pipette off the 80% ethanol and discard.
SECTION: MAS PCR
84. Repeat the previous step.
SECTION: MAS PCR
85. Remove residual 80% ethanol: • Remove the LoBind tube from the magnetic separation rack. • Quick-spin the LoBind tube in a microcentrifuge. • Place the LoBind tube back in a magnetic separation rack until beads separate fully from the solution. Pipette off residual 80% ethanol and discard.
SECTION: MAS PCR
86. Remove the LoBind tube from the magnetic rack. Immediately add 50 µL of elution bufferto each tube and resuspend the beads.
SECTION: MAS PCR
87. Quick-spin the LoBind tube in a microcentrifuge.
SECTION: MAS PCR
88. Incubate at room temperature for 5 minutes to elute DNA.
SECTION: MAS PCR
89. Place the LoBind tube in a magnetic separation rack until beads separate fully from the solution.
SECTION: MAS PCR
90. Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new LoBind tube. Discard old tube with beads.
SECTION: MAS PCR
91. Recommended: Evaluate sample concentration. • Take a 1 µL aliquot from each tube, dilute with 9 µL of elution buffer. Using 1 µL of the dilution, measure DNA concentration with a Qubit fluorometer using the 1x dsDNA HS kit.
SECTION: MAS PCR
92.  Proceed to the next step of the protocol if sample quantity is acceptable (required input: 10 µg). Do not proceed if less than 6 µg is available.
SECTION: MAS Array Formation
93. In a 0.2 mL PCR tube, add 10 µg of sample from Step 4.22, in 47 µL of volume. Dilute with elution buffer going into this step if sample is too concentrated.
SECTION: MAS Array Formation
94. Add 10 µL of MAS enzyme to create single-stranded extensions on PCR-amplified cDNA fragments to enable subsequent directional assembly of 16 PCR products.
SECTION: MAS Array Formation
95. Pipette-mix each sample.
SECTION: MAS Array Formation
96. Run the MAS primer digestion program.
SECTION: MAS Array Formation
97. Add 3 µL of MAS adapter bc01–04 (use a single barcode per sample) and 20 µL of MAS ligation additive to each sample for a total volume of 80 µL.
SECTION: MAS Array Formation
98. Pipette-mix each sample.
SECTION: MAS Array Formation
99. Add the following components in the order and volume listed below to a new microcentrifuge tube.
Adjust component volumes for the number of samples being prepared, plus 10%overage. For
individual preps, add components directly to each sample in the order and volume listed below.
Reaction Mix 3 (RM3): 
 

SECTION: MAS Array Formation
100. Pipette-mix RM3 with wide bore tips.
SECTION: MAS Array Formation
101. Quick-spinRM3 in a microcentrifuge to collect liquid.
SECTION: MAS Array Formation
102. Add 20 µL of RM3 to each sample.
SECTION: MAS Array Formation
103. Pipette-mix each sample with wide bore tips
SECTION: MAS Array Formation
104. Run the MAS array ligation program. Heated lid set at 47°C

 

SECTION: MAS Array Formation
105. Cleanup with 1.2X SMRTbell cleanup beads
SECTION: MAS Array Formation
115. Add 1.2X v/v (120 μL) of resuspended, room-temperature SMRTbell cleanup beads to each sample.
SECTION: MAS Array Formation
116. Pipette-mix the beads with wide bore tips until evenly distributed.
SECTION: MAS Array Formation
117. Quick-spin the tube strip in a microcentrifuge to collect liquid.
SECTION: MAS Array Formation
118. Leave at room temperature for 10 minutes to allow DNA to bind beads.
SECTION: MAS Array Formation
119. Place the tube strip in a magnetic separation rack until beads separate fully from the solution.
SECTION: MAS Array Formation
120. Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant.
SECTION: MAS Array Formation
121. Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into each tube. After 30 seconds, pipette off the 80% ethanol and discard.
SECTION: MAS Array Formation
122. Repeat the previous step.
SECTION: MAS Array Formation
123. Remove residual 80% ethanol: 
Remove the tube strip from the magnetic separation rack.
Quick-spin the tube strip in a microcentrifuge.
Place the tube strip back in a magnetic separation rack until beads separate fully from the
solution.
Pipette off residual 80% ethanol and discard.
SECTION: MAS Array Formation
106. Add 1.2X v/v (120 μL) of resuspended, room-temperature SMRTbell cleanup beads to each sample.
SECTION: MAS Array Formation
107. Pipette-mix the beads with wide bore tips until evenly distributed.
SECTION: MAS Array Formation
108. Quick-spin the tube strip in a microcentrifuge to collect liquid.
SECTION: MAS Array Formation
109. Leave at room temperature for 10 minutes to allow DNA to bind beads.
SECTION: MAS Array Formation
110. Place the tube strip in a magnetic separation rack until beads separate fully from the solution.
SECTION: MAS Array Formation
111. Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant.
SECTION: MAS Array Formation
112. Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into each tube. After 30 seconds, pipette off the 80% ethanol and discard.
SECTION: MAS Array Formation
113. Repeat the previous step.
SECTION: MAS Array Formation
114. Remove residual 80% ethanol: • Remove the tube strip from the magnetic separation rack.
SECTION: MAS Array Formation
124. Remove the tube strip from the magnetic rack. Using a wide bore pipette tip, immediately add 43 µL of elution buffer to each tube and resuspend the beads by pipetting 10 times or until evenly distributed.
SECTION: MAS Array Formation
125. Quick-spin the tube strip in a microcentrifuge to collect liquid.
SECTION: MAS Array Formation
126. Leave at room temperature for 5 minutes to elute DNA.
SECTION: MAS Array Formation
127. Place the tube strip in a magnetic separation rack until beads separate fully from the solution.
SECTION: MAS Array Formation
128. Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new 0.5 mL LoBind tube. Discard old tube strip with beads.
SECTION: MAS Array Formation
129. Recommended: Evaluate sample concentration. 
Take a 1 µL aliquot from each tube, dilute with 4 µL of elution buffer. 
 Measure DNA concentration with a Qubit fluorometer using the 1x dsDNA HS kit. 
The required amount of purified MAS array products to proceed with the DNA damage repair step is 5µg 
SECTION: DNA Damage repair
130. In a 0.2 mL PCR tube, add 5 µg of sample from Step 5.26, in 42 µL of volume. Dilute with elution buffer
going into this step if sample is too concentrated
SECTION: DNA Damage repair
131. Add the following components in the order and volume listed below to a new microcentrifuge tube.
Adjust component volumes for the number of samples being prepared, plus 10% overage. For
individual preps, add components directly to each sample in the order and volume listed below.
Reaction Mix 4 (RM4): 
 

SECTION: DNA Damage repair
132. Pipette-mix RM4.
SECTION: DNA Damage repair
133. Quick-spin RM4 in a microcentrifuge to collect liquid.
SECTION: DNA Damage repair
134. Add 8 µL of RM4 to each sample. Total volume should equal 50 µL.
SECTION: DNA Damage repair
135. Pipette-mix each sample with wide bore tips.
SECTION: DNA Damage repair
136. Quick-spin the strip tube in a microcentrifuge to collect liquid.
SECTION: DNA Damage repair
137. Run the DNA damage repair program. Heated Lid set to 47°C

 

SECTION: DNA Damage repair
138. Clean up with 1.2X SMRTbell cleanup beads
SECTION: DNA Damage repair
139. Add 1.2X v/v (60 μL) of resuspended, room-temperature SMRTbell cleanup beads to each sample.
SECTION: DNA Damage repair
140. Pipette-mix the beads with wide bore tips until evenly distributed.
SECTION: DNA Damage repair
141. Quick-spin the tube strip in a microcentrifuge to collect liquid.
SECTION: DNA Damage repair
142. Leave at room temperature for 10 minutes to allow DNA to bind beads
SECTION: DNA Damage repair
143. Place the tube strip in a magnetic separation rack until beads separate fully from the solution.
SECTION: DNA Damage repair
144. Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant.
SECTION: DNA Damage repair
145. Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into each tube. After 30 seconds, pipette off the 80% ethanol and discard.
SECTION: DNA Damage repair
146. Repeat the previous step.
SECTION: DNA Damage repair
147. Remove residual 80% ethanol: • Remove the tube strip from the magnetic separation rack. • Quick-spin the tube strip in a microcentrifuge. • Place the tube strip back in a magnetic separation rack until beads separate fully from the solution. • Pipette off residual 80% ethanol and discard.
SECTION: DNA Damage repair
148. Remove the tube strip from the magnetic rack. Using a wide bore pipette tip, immediately add 40 µL of elution buffer to each tube and resuspend the beads by pipetting 10 times or until evenly distributed.
SECTION: DNA Damage repair
149. Quick-spin the tube strip in a microcentrifuge to collect liquid.
SECTION: DNA Damage repair
150. Leave at room temperature for 5 minutes to elute DNA.
SECTION: DNA Damage repair
151. Place the tube strip in a magnetic separation rack until beads separate fully from the solution.
SECTION: DNA Damage repair
152. Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new tube strip. Discard old tube strip with bead
SECTION: Nuclease Treatment
153. Add the following components in the order and volume listed below to a new microcentrifuge tube.
Adjust component volumes for the number of samples being prepared, plus 10%overage. For
individual preps, add components directly to each sample from the previous step in the order and
volume listed below.
Reaction Mix 5 (RM5): 
 

SECTION: Nuclease Treatment
154. Pipette-mix RM5.
SECTION: Nuclease Treatment
155. Quick-spin RM5 in a microcentrifuge to collect liquid.
SECTION: Nuclease Treatment
156. Add 10 µL of RM5 to each sample. Total volume should equal 50 µL.
SECTION: Nuclease Treatment
157. Pipette-mix each sample with wide bore tips.
SECTION: Nuclease Treatment
158. Quick-spin the strip tube in a microcentrifuge to collect liquid.
SECTION: Nuclease Treatment
159. Run the nuclease treatment program.
 

SECTION: Final cleanup with SMRTbell cleanup beads
160. Add 60 µL SMRTbell cleanup beads to each sample from the previous step. Using wide bore tips, pipette-mix the beads until evenly distributed.
SECTION: Final cleanup with SMRTbell cleanup beads
161. Quick-spin the tube strip in a microcentrifuge to collect all liquid.
SECTION: Final cleanup with SMRTbell cleanup beads
162. Leave at room temperature for 10 minutes to allow DNA to bind beads.
SECTION: Final cleanup with SMRTbell cleanup beads
163. Place the tube strip in a magnetic separation rack until beads separate fully from the solution.
SECTION: Final cleanup with SMRTbell cleanup beads
164. Slowly pipette off the cleared supernatant without disturbing the beads. It is recommended to save the supernatant in another tube strip in case of poor DNA recovery.
SECTION: Final cleanup with SMRTbell cleanup beads
165. Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into each tube. After 30 seconds, pipette off the 80% ethanol and discard.
SECTION: Final cleanup with SMRTbell cleanup beads
166.  Repeat the previous step.
SECTION: Final cleanup with SMRTbell cleanup beads
167. Remove residual 80% ethanol: • Remove tube strip from the magnetic separation rack. • Quick spin tube strip in a microcentrifuge. • Place tube strip back in a magnetic separation rack until beads separate fully from the solution. Pipette off residual 80% ethanol and discard.
SECTION: Final cleanup with SMRTbell cleanup beads
168. Remove the tube strip from the magnetic rack. Immediately add 20 µL of elution bufferto each tube and resuspend the beads by pipetting 10 times or until evenly distributed with wide bore tips.
SECTION: Final cleanup with SMRTbell cleanup beads
169. Quick-spin the tube strip in a microcentrifuge to collect liquid.
SECTION: Final cleanup with SMRTbell cleanup beads
170.  Leave at room temperature for 5 minutes to elute DNA.
SECTION: Final cleanup with SMRTbell cleanup beads
171.  Place the tube strip in a magnetic separation rack until beads separate fully from the solution.
SECTION: Final cleanup with SMRTbell cleanup beads
172. Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new 0.5 mL LoBind tube. Discard old tube strip with beads.
SECTION: Final cleanup with SMRTbell cleanup beads
173. Take a 1 µL aliquot from each tube. Measure DNA concentration with a Qubit fluorometer using the 1x dsDNA HS kit. Calculate the total mass. Recommended: Further dilute each aliquot to 250 pg/µL with Femto Pulse dilution buffer. Measure final SMRTbell library size distribution with a Femto Pulse system.
SECTION: Final cleanup with SMRTbell cleanup beads
174. Proceed to SMRT Link Sample Setup to prepare the SMRTbell library for sequencing.
SECTION: Final cleanup with SMRTbell cleanup beads
175. Store SMRTbell libraries at 4°C if sequencing within the week. Long-term storage should be at -20°C. Minimize freeze-thaw cycles when handling SMRTbell libraries