TITLE: Ovation RRBS Methyl-seq library prep 
AUTHORS: Nelly Olova

[DESCRIPTION]
This protocol describes a library prep procedure for 1 to 96 samples with the Ovation RRBS Methyl-seq System. The original Tecan Ovation protocol describes a low throughput procedure, therefore this protocol is especially targeted for manual high-throughput preps from 24 to 96 samples, although it can be used for small sample numbers too. Importantly, the Ovation RRBS kit is currently sold as oxBS only (identifying both 5-methyl-C and 5-hydroxymethyl-C), whilst this protocol is modified to exclude the oxidation step and describes a basic RRBS, where both 5mC and 5hmC appear as the same positive signal and are indistinguishable. This protocol has a few further modifications, specifically in the bisulfite conversion step and bead purifications, which have performed better in our hands. 


[BEFORE_START]
Timing suggestions (allow more time until well familiar): 
The calculations can be prepared the day before (Day1).
Sample preparation, MspI digestion, Adaptor ligation - 6 hours with 48 samples; full day with 96 samples (Day 2).
End repair, Purification and Bisulfite conversion - 6 hours (48 samples); full day (96 samples); BS reaction left O/N (Day 3)
Bisulfite-converted DNA desulfonation and purification, library amplification (left O/N) - 6 hours (48 samples); full day (96 samples) (add extra time for optional qPCR) (Day 4)
Amplified library purification and quality control – half a day (48-96 samples); (Day 5)

[GUIDELINES]
Ovation RRBS Methyl-seq library prep (up to 96 samples) 

This protocol is based on Tecan’s Ovation oxBS RRBS protocol, but without the oxidation step, making it suitable for analysing methylation and not hydroxy-methylation. Tecan do not provide a protocol for their 96-sample kit, they have a protocol for 1-16 samples. This protocol is therefore adjusted for a higher-throughput for 24 up to 96 sample preparation.  

Tecan recommend processing not more than 3 strips (24 samples) at a time manually, which means splitting a 96-sample kit in 4 batches. This consideration is mainly because of the washing steps, where timing is crucial and there is a risk of over-drying the beads and reducing yield. This protocol describes a procedure for the washes, which works for manual handling, but does require good pipetting and laboratory experience. The protocol is not designed by Tecan for automated steps (with liquid handling systems), although automation is possible in principle, however, some volumes are too low for most robots. In our personal experience from performing automated wash steps, however, yields can be lower due to loss of beads - a careful manual prep can deliver a 10 to 100-fold higher library yields and much higher quantity consistency between the samples.

Unmethylated DNA control must be purchased separately – this is optional but advisable to include.

[STEPS]
SECTION: Calculations
1. Prepare a list of samples and their concentrations, calculate the required volumes for 100 ng DNA and nuclease-free water for a final reaction volume of 8.5 µL.

 

SECTION: Calculations
2. Calculate the volume of a methylation conversion control to spike in each sample. This is usually unmethylated Lambda virus DNA, added in 1:1000 – 1:5000 ratio to each DNA sample – i.e. up to 0.1 ng unmethylated DNA control per sample. The starting unmethylated DNA stock will be highly concentrated and a dilution will be required.
 

SECTION: Sample Preparation
3. Prepare the unmethylated DNA control – add the necessary total volume to the total volume of water (from the calculations, sum up the unmethylated control DNA and water volumes for all samples), and pre-mix well.

 

SECTION: Sample Preparation
4. Add water (+ unmethylated control) into 0.2 mL PCR strip tubes (if doing 24 samples) or a plate - usually starting with the highest amount of water going down the sample list, column after column. Cross-check for each with the order on the plate schematic, re sample ID, required volume and correct well position.
SECTION: Sample Preparation
5. Add DNA samples in the same sample order, column by column, starting from low to high volume. Cross-check for each with the order on the plate schematic, re sample ID, required DNA volume and correct well position. It is a good practice to cover the completed plate columns as going forward. Keep DNA samples on ice.

SECTION: MspI Digestion
6. Thaw MspI Buffer Mix at Room temperature. Mix MspI Buffer Mix by vortexing, spin and place on ice.  
SECTION: MspI Digestion
7. Spin down the MspI Enzyme Mix and place on ice.

SECTION: MspI Digestion
8. Prepare a master mix by combining MspI Buffer Mix and MspI Enzyme Mix in a 0.2ml PCR tube within an 8-tube PCR strip, according to the volumes shown in Table 1. Mix by pipetting, spin down briefly and immediately place on ice.

 Table 1. MspI Master Mix (advisable extra volume included for high sample number preps).
      REAGENT  1X RXN VOLUME  48X RXN VOLUME (55)  96X RXN VOLUME (110)  STORAGE    MspI BUFFER MIX (BLUE)  1.0 μL  54 μL  110 μL  –20°C     MspI ENZYME MIX (BLUE)  0.5 μL  27 μL  55 μL  –20°C  
 
SECTION: MspI Digestion
9. Distribute equal amounts of master mix to the 8 tubes in the PCR strip for multichannel pipetting.
SECTION: MspI Digestion
10. Add 1.5 µL of MspI Master Mix to each sample tube for a total of 10 µL (with a reliable 0.5-10 μL multichannel and low retention tips). Mix by pipetting (increase pipette volume for the mixing), spin down and place on ice.

SECTION: MspI Digestion
11. Place the tubes in a thermal cycler programmed to run Program 1 (MspI Digestion):
 37 °C – 60 min, 
hold at 4 °C.
SECTION: MspI Digestion
12. In the meantime take out to thaw at RT the water and buffers for adapter ligation – see Adaptor Ligation.
SECTION: MspI Digestion
13. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice. 
 

SECTION: Adaptor Ligation
14. Thaw Ligation Buffer Mix L1 at Room temperature. Mix well by vortexing, spin and place on ice.

SECTION: Adaptor Ligation
15. Thaw the Ligation Adaptor Plate on ice, spin down, and return to ice. 
 


SECTION: Adaptor Ligation
16. Spin down the Ligation Enzyme Mix L3 and place on ice or in a cooling rack. 


SECTION: Adaptor Ligation
17. Pierce the Ligation Adaptor Plate seal with the tips and aspirate 3 µL (the entire amount) of the Ligation Adaptor Mix L2 (with a 0.5-10ul multichannel and low retention tips). Add one adaptor barcode per sample, noting the adaptor plate positions per each sample. Mix thoroughly by pipetting and keep on ice.

SECTION: Adaptor Ligation
18. Just prior to use, prepare a master mix by combining D1, L1 and L3, according to the volumes shown in Table 2. Mix by pipetting slowly, without introducing bubbles, spin and place on ice. Use the master mix immediately.


 


Table 2. Ligation Master Mix (advisable extra volume included for high sample number preps)
      REAGENT  1X RXN VOLUME  48X RXN VOLUME (55)  96X RXN VOLUME (105)  STORAGE    NUCLEASE-FREE WATER (GREEN: D1)  2.0 μL  110 μL  210 μL  -    LIGATION BUFFER MIX (YELLOW: L1)  4.0 μL  220 μL  420 μL  –20°C     LIGATION ENZYME MIX (YELLOW: L3)  1.0 μL  55 μL  105 μL  –20°C  
 
SECTION: Adaptor Ligation
19. Add 7 µL Ligation Master Mix to each reaction tube for a total of 20 µL (with a multi-dispenser or a reliable 0.5-10μl/2-20μl multichannel and low retention tips). Mix thoroughly by pipetting slowly and gently, spin down and place on ice. Proceed immediately with the incubation.
SECTION: Adaptor Ligation
20. Place the tubes in a thermal cycler programmed to run Program 2 (Ligation): 
25 °C – 30 min, 
70 °C – 10 min, 
hold at 4 °C.
SECTION: Adaptor Ligation
21. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice. 
 

SECTION: Final Repair
22. Remove the Magnetic Bead Solution and Binding Buffer 1 from 4 °C and place at Room temperature for use in the next step.
SECTION: Final Repair
23. Thaw Final Repair Buffer Mix (FR1) at Room temperature. Mix by vortexing, spin down and place on ice.

SECTION: Final Repair
24. Spin down Final Repair Buffer Enzyme (FR2) and place on ice.
SECTION: Final Repair
25. Prepare a master mix by combining FR1, FR2 and Nuclease-free Water (D1) according to the volumes shown in Table 3.

Table. 3 Final Repair Master Mix  (advisable extra volume included for high sample number preps) 
      REAGENT  1X RXN VOLUME  48X RXN VOLUME (54)  96X RXN VOLUME (100)  STORAGE    FINAL REPAIR BUFFER MIX (PURPLE: FR1 ver   4)  6 μL  324 μL  600 μL  –20 °C    FINAL REPAIR ENZYME MIX (PURPLE: FR2)  0.5 μL  27 μL  50 μL  –20 °C     NUCLEASE-FREE WATER (GREEN: D1)  13.5 μL  729 μL  1350 μL  –  
 
SECTION: Final Repair
26. Add 20 µL of the Final Repair Master Mix to each sample for a total of 40 µL (use a reliable multichannel pipette and low retention tips). Mix by pipetting each time, spin down and place on ice.
SECTION: Final Repair
27. Place the tubes in a thermal cycler pre-heated to 60 °C (if possible) and programmed to run Program 3 (Final Repair): 
60 °C – 10 min, 
70 °C – 10 min, 
hold at 4 °C.
SECTION: Final Repair
28. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice. 

 

SECTION: DNA Purification
29. Remove Magnetic Bead Solution and Binding Buffer 1 from storage and place on bench top. Ensure they have reached Room temperature before use.

 

SECTION: DNA Purification
37. Prepare a fresh stock of 80% ethanol, using the Ultra Pure water provided with the kit. Mix by inversion and place at Room temperature. 
 

SECTION: DNA Purification
30. Once warmed to room temperature, mix Binding Buffer 1 by inversion until homogenized.
SECTION: DNA Purification
31. Vortex Magnetic Bead Solution until homogenized.
SECTION: DNA Purification
32. Prepare a master mix of Magnetic Bead Binding Solution 1 (MBBS1) as directed in Table 4. Binding
Buffer 1 is supplied in 5.4 mL bottles suitable for a 48 sample mix, use two bottles for a full plate.

 

Table. 4 Magnetic Bead Binding Solution 1 Master Mix. 
      REAGENT  1X RXN VOLUME  48X RXN VOLUME (55)  96X RXN VOLUME (105)  STORAGE    BINDING BUFFER 1  100 μL  5.5 mL  10.5 mL  4 °C     MAGNETIC BEAD SOLUTION  2.0 μL  110 μL  210 μL  4 °C  
 

SECTION: DNA Purification
33. Vortex MBBS1 master mix thoroughly to ensure the beads are homogenized in solution.
SECTION: DNA Purification
34. At Room temperature, add 10 µL of Ultra Pure water to each sample for a total of 50 µL (with a reliable 0.5-10 μl/2-20 μl multichannel and low retention tips or an Eppendorf dispenser, without touching the wells, 1.0 ml tip).
SECTION: DNA Purification
35. Add 100 µL of MBBS1 master mix to each 0.2 mL tube/well containing 50 µL sample for a total of 150 µL (with a dispenser, 2-10 ml tip). Mix by pipetting with a multichannel pipette set at 130 μL and centrifuge briefly.
SECTION: DNA Purification
36. Incubate at Room temperature for 20 min (can be longer if a break is needed).
SECTION: DNA Purification
38. Transfer tubes/plate to a magnetic separation plate and incubate at Room temperature for 10 min or until the solution of beads is completely clear. 

SECTION: DNA Purification
39. Keeping the tubes on the magnet, carefully remove the supernatant and discard it – with a manual multichannel pipette set at 140 µL.
SECTION: DNA Purification
40. With the tubes/plate still on the magnet, carefully add 200 µL of 80% ethanol wash to the wells without disturbing the bead pellet (with a dispensing multi-channel set at 1200 µL, 6x strips/half a plate at a time).
SECTION: DNA Purification
41. Remove and discard the 200 µL 80% ethanol wash, avoiding aspiration of the bead pellet (with a manual 20-200 μL multichannel, regular tips). Complete this step as quickly as possible, ideally within 2 minutes. 

SECTION: DNA Purification
42. Repeat Steps 40 and 41 to perform 2 x 200 µL 80% ethanol washes in total. Remove as much of the final wash as possible – first with a 20-200 μL tip, and a second time with a 10/20 μL
tip if necessary.
SECTION: DNA Purification
43. Air dry the bead pellets for 5-10 minutes at Room temperature. 
 

SECTION: DNA Purification
44. Remove the tubes from the magnet.
SECTION: DNA Purification
45. Add 11 µL of Elution Buffer directly onto the bead pellet. 
 

SECTION: DNA Purification
47. Incubate at Room temperature for at least 5 min to elute the TrueMethyl converted DNA from the beads.
SECTION: DNA Purification
48. Seal the plate and centrifuge briefly to collect all drops at the bottom of the tubes.
SECTION: DNA Purification
49. Transfer tubes/plate to a magnetic separation plate and incubate at Room temperature for 5-10 minutes or until the solution of beads is completely clear.
SECTION: DNA Purification
50. Removing the seal one column/strip at a time, carefully aspirate 10 µL of the eluate, ensuring as few beads as possible are carried over, and transfer to a new microcentrifuge plate/tubes (use a 0.5-10uL multichannel with low retention tips). Keep at Room temperature if continuing with section Bisulfite Conversion. Can store in fridge or freezer if an 5 min break is needed.


 

SECTION: Bisulfite Conversion
51. Set a heat block or heated orbital incubator to 70 °C. 

SECTION: Bisulfite Conversion
52. Remove Bisulfite Diluent and Bisulfite Reagent aliquots from storage and place on bench top. Remove 1 aliquot of Bisulfite Reagent for every 25 reactions to be processed and spin quickly to remove any powder from cap (i.e. a full 96-well plate uses 4 Bisulfite Reagent vials).
SECTION: Bisulfite Conversion
53. Prepare Bisulfite Reagent Solution by adding 700 µL of Bisulfite Diluent to each aliquot of Bisulfite Reagent.
 

SECTION: Bisulfite Conversion
54. Seal the lid of each aliquot with Bisulfite Reagent Solution tightly.
SECTION: Bisulfite Conversion
55. Incubate the aliquots of Bisulfite Reagent Solution for minimum 30 min at 70 °C and vortex regularly until the Bisulfite Reagent Solution is completely (or nearly completely) dissolved. This step can take up to 180 min for the reagent to dissolve.

SECTION: Bisulfite Conversion
56. Spin down Bisulfite Reagent Solution briefly and place at Room temperature.

SECTION: Bisulfite Conversion
57. Ensure purified DNA samples from previous step are at Room temperature before proceeding.

SECTION: Bisulfite Conversion
58. Prepare Bisulfite Conversion Reaction mix by adding 30 µL of Bisulfite Reagent Solution to each 10 µL of DNA for a total of 40 µL (with multi-dispenser, 1 mL tips).
SECTION: Bisulfite Conversion
59. Mix by pipetting with a 20-200 μl multichannel, spin down and place at Room temperature. 

SECTION: Bisulfite Conversion
60. Place the tubes in a pre-warmed thermal cycler programmed to run Program 5 (Bisulfite Conversion):
 99 °C – 10 min, 
65 °C – 105 min, 
hold at 20 °C.
 

SECTION: Bisulfite Conversion
61. Once the bisulfite conversion is complete, centrifuge samples briefly to collect the solution at bottom of the tubes.
SECTION: Bisulfite Conversion
62. Continue to Bisulfite-Converted DNA Desulfonation and Purification.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
63. Remove Desulfonation Buffer, Binding Buffer 2, Magnetic Bead Solution and Elution Buffer from storage and place at Room temperature for a minimum of 30 min before use.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
64. Prepare a fresh stock of 80% Ethanol. Mix by vortexing or inversion.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
65. Mix Binding Buffer 2 by inversion until homogenized.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
66. Vortex Magnetic Bead Solution until homogenized.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
67. Prepare a master mix of Magnetic Bead Binding Solution 2 (MBBS2) as directed in Table 5. Each kit BB2 bottle is filled with exactly 9 mL solution ready to use for a 48 sample prep; use two bottles for a full plate.

Table. 5 Magnetic Bead Binding Solution 2 Master Mix.
 
      REAGENT  1X RXN VOLUME  48X RXN VOLUME  96X RXN VOLUME  STORAGE    BINDING BUFFER 2  160 μL (200)  9 mL  18 mL  4 °C     MAGNETIC BEAD SOLUTION  1.92 μL (2.4)  108 μL  216 μL  4 °C  
  
 

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
68. Vortex MBBS2 thoroughly to ensure the solution is homogenous before aliquoting.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
69. Carefully add 160 µL of MBBS2 to each tube containing 40 µL of bisulfite converted sample for a total of 200 µL (with multi-dispenser, 5 mL tip). Mix thoroughly by pipetting slowly and gently, spin down and place at Room temperature.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
70. Incubate at Room temperature for 30 min to 60 min, vortexing if necessary, or alternating plate position by rotation upwards-downwards after sealing VERY WELL.

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
71. Prepare desulfonation buffer by adding 7 mL 100% ethanol to 3 mL of Desulfonation Buffer for up to 48 samples, or 14 mL EtOH + 6 mL Desulfonation buffer for up to 96 samples, respectively.

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
72. Centrifuge briefly to collect the solution at the bottom of the tubes/wells. To bring down the beads from the liquid remaining above the magnet, centrifuge at 2200 rcf / 4700 rpm, 2 min.

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
73. Place the tubes/plate onto the magnet and incubate at Room temperature for 10-15 minutes to completely clear the solution of beads.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
74. Carefully remove the supernatant and discard it, without discarding any beads.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
75. Add 200 µL of 80% Ethanol to each sample tube/well (with a dispensing multichannel up to 1200 µL), 6 columns at a time). 
 

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
76. Carefully remove the 80% Ethanol wash and discard it. Remove as much of the wash as possible.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
77. Add 200 µL of Desulfonation Buffer with added Ethanol onto the bead pellet. Incubate at Room temperature for 5 min. 
 

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
78. Carefully remove 200 µL of the Desulfonation Buffer and discard it. Remove as much of the Desulfonation Buffer as possible without disturbing the bead pellet.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
79. With the tubes/plate still on the magnet, add 200 µL of 80% Ethanol to each sample tube without disturbing the bead pellet and incubate for 30 s (with a dispensing multichannel set at 1200 μL, 6x strips/half a plate at a time).

 

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
80. Remove the 200 µL 80% Ethanol wash and discard it, avoiding aspiration of the bead pellet (with a manual 20-200 μL multichannel, regular tips). This step must be completed as quickly as possible (ideally within 2 min)

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
81. Repeat steps 79–80 to perform 2 x 200 µL 80% Ethanol washes in total. Remove as much of the final wash as possible – first with a 20-200 μL tip, and a second time with a 10/20 μL tip, if necessary. 
 

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
82. Air-dry the the beads on the magnet for 5-10 minutes. Inspect each tube carefully to ensure that all of the Ethanol has evaporated – the pellet should be non-shiny (matt look) when it is ready. Using a pipette with 10 μl tips can help with aspirating Ethanol drops while still on the magnet.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
83. Remove the tubes/plate from the magnet.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
84. Quickly add Elution Buffer with a multi-dispensing pipette aiming directly onto the bead pellet: 
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
85. Mix and resuspend completely the beads in the Elution Buffer with a multichannel pipette pipette – make sure all beads are removed from the well walls and brought in solution. 
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
87. Seal the plate and centrifuge briefly to collect the samples at the bottom of the tubes/wells.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
88. Place the tubes/plate onto the magnet and incubate at Room temperature for 5-10 minutes to completely clear the solution of beads.

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
89. Opening the seal one column/strip at a time, carefully transfer eluate into a fresh 0.2 mL PCR plate: 
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
84.1. For Library Amplification Optimzation with qPCR (recommended), resuspend the beads in 25 µL of Elution Buffer.
SECTION: Bisulfite-Converted DNA Desulfonation and Purification
84.2.  If qPCR optimization is not required, resuspend the beads in 21 µL of Elution Buffer.

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
89.1. For Library Amplification Optimzation with qPCR (recommended first time only), transfer 24 µL of eluate.

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
89.2.  If qPCR optimization is not required, transfer 20 µL of eluate.


 

SECTION: Library Amplification Optimization with qPCR (optional)
90. 
 
Prepare a master mix by combining P2, P3 and 20x EvaGreen in an appropriately sized capped tube according to the volumes shown in Table 6. Add P3 at the last moment and mix well by pipetting, taking care to avoid bubbles. Spin down and place on ice.  

Table. 6 Library Amplification qPCR Master Mix (with extra volumes for high sample number). 
      REAGENT  1X RXN VOLUME  4X RXN VOLUME  96X RXN VOLUME  STORAGE    AMPLIFICATION PRIMER MIX (RED: P2 ver 8)  1.0 μL  4.2 μL  100 μL  –20 °C     AMPLIFICATION ENZYME MIX (RED: P3 ver 3)  4.5 μL  18.9 μL  450 μL  –20 °C    20X EvaGreen  0.5 μL  2.1 μL  50 μL  –20 °C  
 
SECTION: Library Amplification Optimization with qPCR (optional)
91. Aliquot 6 µL of master mix per sample into an appropriate optically clear PCR plate. Spin down and place on ice.

SECTION: Library Amplification Optimization with qPCR (optional)
92. on ice, add 4 µL of sample to each 6 µL of Library Amplification qPCR Master Mix for a total of 10 µL per reaction. Keep the remaining 20 µL of sample on ice.
SECTION: Library Amplification Optimization with qPCR (optional)
93. Mix well by pipetting, spin down and place on ice.
SECTION: Library Amplification Optimization with qPCR (optional)
94. Perform qPCR with the following cycling conditions: 
95 °C – 2 min, 
35 x (95 °C– 15 s, 60 °C – 1 min, 72 °C – 30 s).

SECTION: Library Amplification Optimization with qPCR (optional)
95. Examine the log fluorescence vs. cycle number plot from the qPCR system to determine the appropriate number of library amplification cycles. Select a cycle number within the middle to late exponential phase of the amplification plot. Examples are provided in Figure 8 of the original Tecan kit manual.
SECTION: Library Amplification
96. Remove Agencourt Beads from 4 °C and DR1 from -20 °C and place at Room temperature for use in the next step.
SECTION: Library Amplification
97. Spin down Amplification Enzyme Mix (P3) and place on ice.

SECTION: Library Amplification
98. Thaw Amplification Primer Mix (P2) at Room temperature. Mix by vortexing, spin down and place on ice.

SECTION: Library Amplification
99. Prepare a master mix by combining P2 and P3 according to the volumes shown in Table 7. Mix well by pipetting, taking care to avoid bubbles, spin down and place on ice.

Table. 7 Library Amplification Master Mix (with extra volumes for high sample number). 
      REAGENT  1X RXN VOLUME  48X RXN VOLUME (50)  96X RXN VOLUME (100)  STORAGE    AMPLIFICATION PRIMER MIX (RED: P2 ver 8)  5.0 μL  275 μL  500 μL  –20 °C     AMPLIFICATION ENZYME MIX (RED: P3 ver 3)  25.0 μL  1.375 μL  2.500 μL  –20 °C  
 
SECTION: Library Amplification
100. on ice, add 30 µL of Amplification Master Mix to each sample for a total of 50 µL.

SECTION: Library Amplification
101. Place tubes in a pre-warmed thermal cycler programmed to run Program 6 (Library Amplification): 
95 °C – 2 min,
N(95 °C – 15 s,
60 °C – 1 min,
72 °C – 30 s),
 72 °C – 5 min, 
hold at 10 °C.
SECTION: Library Amplification
102. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice. 
 

SECTION: Amplified Library Purification
103. 
Ensure the Agencourt beads and DR1 Resuspension Buffer have reached Room temperature before proceeding.
SECTION: Amplified Library Purification
104. Resuspend the beads by inverting and tapping the tube. Ensure the beads are fully resuspended before adding to samples. After resuspending, do not spin the beads.
SECTION: Amplified Library Purification
105. Add 50 µL (1 volume) of the bead suspension to each reaction. Mix thoroughly by pipetting 10 times.

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

SECTION: Amplified Library Purification
107. Prepare fresh 80% EtOH solution in a 50 mL falcon tube – 40 mL for a full plate, 20 mL for a half plate.
SECTION: Amplified Library Purification
108. Transfer the tubes to the magnet and let stand 5 min to completely clear the solution of beads.

SECTION: Amplified Library Purification
109. If doing your own QC, prepare a plate for Tapestation aliquots and take out Tapestation kit reagents to equilibrate to Room temperature (see Library Quality Control step).

SECTION: Amplified Library Purification
110. Carefully remove 90 µL of the binding buffer and discard it. Leaving some of the volume behind minimizes bead loss at this step. 

 

SECTION: Amplified Library Purification
111. With samples still on the magnet, add 200 µL of of 80% ethanol to each sample tube and incubate for 30 s (with a dispensing multichannel set at 1200 μL, 6x strips/half a plate at a time).


 

SECTION: Amplified Library Purification
112. Remove the 200 μL of 80% Ethanol wash and discard it (with a manual 20-200 μL multichannel, regular tips). This step must be completed as quickly as possible (ideally within 2 minutes). 


SECTION: Amplified Library Purification
113. Repeat steps 111 and 112 for a total of two washes. Remove all remaining traces of ethanol after the second wash. 

 

SECTION: Amplified Library Purification
114. Air dry the beads on the magnet for 5-10 minutes. Inspect each tube carefully to ensure that all of the ethanol has evaporated. It is critical that all residual ethanol be removed prior to continuing, but it is also critical to not let beads over-dry as this reduces yield (they have over-dried when they crack).
SECTION: Amplified Library Purification
115. Remove the tubes from the magnet and add 20 µL DR1 Resuspension buffer to the dried beads (with dispenser aiming at the beads). 
SECTION: Amplified Library Purification
116. Mix thoroughly with a multichannel pipette, set at 10 µL, to ensure all beads are resuspended – if the beads have cracked this may require mixing for a few minutes. Take any beads remaining on the sides and make sure all beads are covered in solution.
SECTION: Amplified Library Purification
117. Seal the plate/close the tubes and centrifuge briefly to collect sample at bottom of the tubes/wells. 
SECTION: Amplified Library Purification
119. Carefully remove the seal one column/strip at a time, and aspirate 19 µL of the eluate, ensuring as few beads as possible are carried over, and transfer to a fresh plate / set of PCR tubes. Keep the eluate on ice. 
 

SECTION: Amplified Library Purification
120. Once the elution is complete, proceed with preparing library aliquots for a Tapestation QC run in the next section Library Quality Control (QC). If QC is not planned soon, seal the library plate well and store at -20 °C . 

SECTION: Prepare Library Aliquots For QC
121. Aliquot 1 µL of nuclease-free water in the wells of a 96-well plate, matching the number and positions of eluted libraries (this is for a 1 in 2 dilution, respectively, higher dilutions can also be used, depending on QC equipment sensitivity). 

 


SECTION: Prepare Library Aliquots For QC
122. From the elution plate from Step 120, take 1 µL of purified library with a multichannel pipette, transfer to the QC plate with water and mix.
SECTION: Prepare Library Aliquots For QC
124. If performing a Tapestation run, continue with the High Sensitivity DNA 1000 Tapestation kit protocol (procedure not described here). If taking the DNA to be measured elsewhere, seal the QC plate and take to the facility. The QC plate can be stored at -20 °C, although evaporation can occur during long term storage.

SECTION: Prepare Library Aliquots For QC
123. The plate with purified libraries can now be sealed and stored long term at -20 °C.


SECTION: DNA Purification
46. Mix thoroughly with a 0.5-10μl multichannel to ensure all beads are resuspended. Don’t centrifuge if there are drops on the walls!

 

SECTION: Bisulfite-Converted DNA Desulfonation and Purification
86. Incubate at Room temperature for 5 min to elute the bisulfite converted DNA from the beads.

SECTION: Amplified Library Purification
118. Transfer the tubes to the magnet and let the samples stand for 5 min or until the solution is completely clear of beads.