TITLE: scNMT-seq v2
AUTHORS: Liyun Zhao, Thomas Lefevre, Thierry Voet, Bernard Thienpont

[DESCRIPTION]
scNMT-seq (single cell Nucleosome, Methylome, and Transcriptome sequencing) allows the parallel study of a single cell chromatin status, methylation profile, and transcriptome.

Here, we are developing and testing modifications of the scNMT-seq pipeline. The protocol is carried out in 96w plates and typically takes 4-5 days to complete.
The number of pre-amplification cycles is adjusted to tackle the problem of poor recovery after BS conversion.Primers are optimized for first-strand and second-strand synthesis to solve the problem of unmapped reads and poor amplification. Both are testified as compatible with theoriginal the original scNMTseq.

[STEPS]
SECTION: Cell isolation and GpC methylation
1. 
Prepare plates containing 2.5µl GpC methylase reaction mixture in each well: 
 
 
     Component (Cinitial)      Cfinal      Volume       Mastermix (     samples)        M.CviPI reaction buffer (10x)      1x      0.25 µl               M.CviPI (4U/µl)      2U      0.5 µl               SAM (320µM)      160µM      1.25 µl               IGEPAL (10%)      0.1%      0.025 µl               RNasein (20U/µl)      1U/µl      0.125µl               Nuclease-free water             0.35 µl          
 

SECTION: Cell isolation and GpC methylation
2. 
Isolate cells manually or using FACS in 2.5 µLof GpC methylase reaction buffer
SECTION: Cell isolation and GpC methylation
3. 
After cell isolation, spin down plates at ≥1000g for ≥10s 4 °C 

SECTION: Cell isolation and GpC methylation
4. 
Incubate the samples at 37 °C for 15 min 

SECTION: Cell isolation and GpC methylation
5. 
Stop reaction by adding 5 µLRLT plus buffer
SECTION: Cell isolation and GpC methylation
6. 
Stoe the plates at-80 °C until processed

SECTION: Oligo-dT30VN bead preparation
7. 
Add55 µLDynabeads into a new Eppendorf tube. Place the tube on a magnet for 2 min and discard supernatant
SECTION: Oligo-dT30VN bead preparation
8. 
Resuspend beads in 200 µLDynabead solution A  (0.1 Molarity (M) NaOH, 0.05 Molarity (M) NaCl). Place the tube on a magnet for 2 min and discard supernatant

SECTION: Oligo-dT30VN bead preparation
9. 
Repeat step 8 once

SECTION: Oligo-dT30VN bead preparation
10. 
Resuspend beads in 200 µL Dynabead solution B (0.1 Molarity (M) NaCl). Place on a magnet for 2 min and discard supernatant

SECTION: Oligo-dT30VN bead preparation
11. 
Resuspend the beads in55 µLof 2x B&W (2 Molarity (M) NaCl, 10 millimolar (mM) Tris-HCl, 1 millimolar (mM) EDTA) and 55 µL Biotinylated Oligo-dT30VN (100 micromolar (µM)).
Incubate 20 min on a thermomixer while shaking at 2000rpm at Room temperature

In the meantime, prepare the bead resuspension buffer
 
     Superscript FS buffer (5x)      110µl        Nuclease-free Water      412.5µl        RNase inhibitor (20U/µl)      27.5µl   
 
In the meantime, prepare 1x B&W buffer by mixing 440 µL Nuclease-free water with 440 µL 2x B&W buffer
SECTION: Oligo-dT30VN bead preparation
12. 
Place beads on a magnet for2 min and discard supernatant
SECTION: Oligo-dT30VN bead preparation
13. 
Resuspend the beads in 200 µL 1x B&W buffer. Place beads on a magnet for 2 min and discard supernatant
SECTION: Oligo-dT30VN bead preparation
14. 
Repeat step 13 three more times
SECTION: Oligo-dT30VN bead preparation
15. 
Resuspend the beads in the bead resuspension buffer 
SECTION: Physical separation of mRNA and gDNA
16. 
Thaw the 96-well plate containing the single cell lysates on ice
SECTION: Physical separation of mRNA and gDNA
17. 
Add 1 µL ERCC spike-ins at 1:1Million - 1:128Million dilution to each sample using a multi-dispensing pipette. Run the pulse centrifugation program to spin ERCCs down to the bottom 
SECTION: Physical separation of mRNA and gDNA
18. 
Take 4 tubes(1069 µLper tube ) of G&T wash buffer(50 millimolar (mM) Tris–HClpH 8.3, 75 millimolar (mM) KCl, 3 millimolar (mM) MgCl2, 0.5% Tween 20 Solution) and add to each tube137.5 µL DTT and 25 µL RNaseIn

SECTION: Physical separation of mRNA and gDNA
19. 
Add 50 µL G&T-Seqwash buffer per well to the“G&T-Seq wash plate”
SECTION: Physical separation of mRNA and gDNA
22. 
Spin all plates and run the adapted G&T-separation program robotically or manually.



While the separation program is running, prepare the RT master mix
     Component (Cin)      Cfinal      Volume      Mastermix ( samples)        dNTP (10mM)      1mM      0.5µl               TSO (100µM)      1µM      0.05µl               MgCl2 (1M)      6mM      0.03µl               Betain (5M)      1M      1µl               S II First strand buffer (5x)      1x      1µl               DTT (100mM)      5mM      0.25µl               Nuclease-free water             1.8µl               RNase inh (20U/µl)      0.5U/µl      0.125µl               Superscript reverse transcriptase II   (200U/µl)      10U/µl      0.25µl          

 

SECTION: Physical separation of mRNA and gDNA
20. 
Add 10 µL Oligo-dT beads per well to the“bead plate”
SECTION: Physical separation of mRNA and gDNA
21. 
Add an empty non-skirted 96 well plate labeled “gDNA collection”
SECTION: Reverse transcription
23. 
Collect the polyA(+) mRNA plate and using the multi-dispenser dispense 5 µLRT master mix into each well of the bead-containing 96-well plate
SECTION: Reverse transcription
24. 
Seal the mRNA and gDNA plates and spin. 

Store gDNA at -80 °C until processed

SECTION: Reverse transcription
25. 
Incubate the polyA(+) mRNA 96-well plate on a thermomixer C using the program below (approx. duration 105 min)
     Cycle      Temp   (°C)      Time      Mixing   (rpm)        1      42      2 min      2000        2      42      60 min      1500        3      50      30 min      1500        4      60      10 min      1500   

SECTION: Reverse transcription
26. 
In the meantime prepare PCR mastermix
     Component      Volume      Mastermix (     samples)        KAPA   HiFi HotStart ReadyMix (2x)      6.25µl               IS   PCR primer (10µM)      0.124µl               Nuclease-free   water      1.13µl          

 
SECTION: PCR amplification of cDNA
27. 
Add 7.5 µLPCR reaction mastermix, seal the plate and centrifuge

SECTION: PCR amplification of cDNA
28. 
Resuspend the beads for30 s at 2000rpm using the Thermomixer

SECTION: PCR amplification of cDNA
29. 
Perform cDNA amplification as follows
 
     Cycles      Temperature (°C)      Time        1      98      3 min          98      20 s        67      15 s         72      6 min        1      72      5 min        1      4      Hold   
 
SECTION: PCR cleanup of amplified cDNA
30. 
Add 12.5 µL Agencourt AMPure beads (1:1 ratio), mix thoroughly by pipetting up and down

SECTION: PCR cleanup of amplified cDNA
31. 
Incubate5 min at Room temperature 

SECTION: PCR cleanup of amplified cDNA
32. 
Pellet the beads on a Low-elution magnet for 5 min

SECTION: PCR cleanup of amplified cDNA
33. 
Remove the supernatant without disturbing the beads

SECTION: PCR cleanup of amplified cDNA
34. 
Wash the beads twice with 150 µL of freshly prepared 80% ethanol for 10 s

SECTION: PCR cleanup of amplified cDNA
35. 
Allow the beads to dry for approximately 5 min . Resuspend in25 µL nuclease-free water. Incubate for 2 min Room temperature 

SECTION: PCR cleanup of amplified cDNA
36. 
Return the 96-well plate to the magnet and allow the Agencourt AMPure beads to settle for 5 min

SECTION: PCR cleanup of amplified cDNA
37. 
Carefully transfer the supernatant to a new 96-well plate 

 

SECTION: Library preparation of cDNA (Nextera XT)
38. 
Dilute the cDNA of each sample to 0.2ng/μl with nuclease-free water
SECTION: Library preparation of cDNA (Nextera XT)
39. 
Add 2.5 µL Tagment DNA(TD) buffer to a new Hard-Shell skirted 96-well plate 



SECTION: Library preparation of cDNA (Nextera XT)
40. 
Add 1.25 µL diluted cDNA and 1.25 µL amplicon tagment mix (ATM) to TD buffer and mix
SECTION: Library preparation of cDNA (Nextera XT)
41. 
Centrifuge the plate at 280 x g, 1 min, 20 °C 

SECTION: Library preparation of cDNA (Nextera XT)
42. 
Incubate on a thermal cycler 
 
     Segment      Temp   (°C)      Duration   (min)        1      55      10        2      10      Hold   
 
SECTION: Library preparation of cDNA (Nextera XT)
48. 
Centrifuge the samples at 280 x g, 1 min, 20 °C  and amplify as follows: 

 
     Cycle      Temp   (°C)      Duration           1      72      3min        2      95      30s        3-14      95      10s        55      30s        72      30s        15      72      5min        16      4      Hold   
 

SECTION: Library preparation of cDNA (Nextera XT)
43. 
Add 1.25 µLNeutralize Tagment Buffer (NT) 
SECTION: Library preparation of cDNA (Nextera XT)
44. 
Vortex & spin down at  800 x g, 1 min, 20 °C

SECTION: Library preparation of cDNA (Nextera XT)
45. 
Incubate at Room temperature 5 min

SECTION: Library preparation of cDNA (Nextera XT)
46. 
Add 1.25 µL Index (i7) adapter to each column and 1.25 µL Index 2 (i5) adapter to each row 
SECTION: Library preparation of cDNA (Nextera XT)
47. 
Add3.75 µL Nextera PCR mastermix and mix 
SECTION: Library preparation of cDNA (Nextera XT)
49. 
Centrifuge the plate at 280 x g, 1 min, 20 °C

SECTION: Library preparation of cDNA (Nextera XT)
50. 
Purify libraries at a 0.66:1 ratio and elute in 12.5 µL EB buffer

 

SECTION: Library preparation of cDNA (Nextera XT)
51. 
Pool libraries and quantify using qPCR
 

SECTION: scBS-seq library preparation (gDNA)
52. 
SECTION: Bisulfite conversion
53. 
Prepare the CT conversion reagent by mixing 7.9 mL M-Solubilisation buffer and 3 mLM-Dilution buffer and 15 min vortexing at Room temperature 
Finally,add1.6 mLM-Reaction buffer and vortex4 min at Room temperature 




SECTION: Bisulfite conversion
54. 
Add 32.5 µL AMPure XP beads to the gDNA plate (0.65:1 ratio)
SECTION: Bisulfite conversion
55. 
Incubate20 min Room temperature 

SECTION: Bisulfite conversion
56. 
Place the plate on the magnet for 20 min and discard the supernatant

SECTION: Bisulfite conversion
57. 
Wash the beads twice with 200 µL80% ethanol

SECTION: Bisulfite conversion
58. 
Resuspend the beads in10 µL elution buffer, optionally containing 60fg unmethylated lambda DNA

 


SECTION: Bisulfite conversion
59. 
Add 65 µL CT conversion reagent without mixing

 



SECTION: Bisulfite conversion
60. 
Incubate the mixture as follows:
  
     Segment      Temperature   (ºC)      Duration   (min)        1      98      8        2      65      180        3      4      Hold   
 
 

SECTION: Purification of the bisulfite converted DNA
61. 
Mix 300 µL M-binding buffer and 5 µL MagBinding beads
 



SECTION: Purification of the bisulfite converted DNA
62. 
Transfer the samples to the M-binding buffer - MagBinding beads mix and incubate 5 min Room temperature 


SECTION: Purification of the bisulfite converted DNA
63. 
Pellet the beads on a magnet for 3 minand discard the supernatant 

SECTION: Purification of the bisulfite converted DNA
64. 
Resuspend the beads in200 µL M-Wash buffer 
SECTION: Purification of the bisulfite converted DNA
65. 
Pellet beads on the magnet and discard the supernatant. Resuspend the beads in 100 µLM-Desulphonation buffer and incubate 15 minRoom temperature 

 


SECTION: Purification of the bisulfite converted DNA
66. 
Pellet beads on the magnet and discard the supernatant. Wash the beads twice with 200 µL M-Wash buffer 
SECTION: Purification of the bisulfite converted DNA
67. 
Dry the beads on a heating element at 55 °C for 10 min 
In the meantime, prepare the pre-amplification mix as follows
 
 
     Component      Amount (µl)      Final concentration      Mastermix (     samples)        Blue buffer (10×)      4      1x               dNTP mix (10mM)      1.6      0.4mM               Preamp Oligo (10 µM)      1.6      O.4 µM               H2O      32.8                      Total volume      40                 
 
SECTION: Pre-amplification
68. 
Resuspend the beads in a 40 µLpre-amplification mix
SECTION: Pre-amplification
69. 
Incubate the mixture at 55 °C for 4 min and place it on the magnet

SECTION: Pre-amplification
70. 
After the beads are pelleted transfer 39 µL to a new plate
SECTION: Pre-amplification
71. 
Incubate the samples 3 min at65 °C and immediately cool on a pre-cooled aluminum rack
Centrifuge the plate at 500 x g, 10 s 

SECTION: Pre-amplification
72. 
Add 1 µL klenow exo- polymerase (50U/µl)
Vortex the samples and amplify as follows:
 
     Segment      Temp (ºC)      Duration (min)      Ramp speed (ºC/min)        1      4      5      -        2      4-37      8.25      4        3      37      30      -        4      4      Hold          
 
SECTION: Pre-amplification
73. 
In the meantime, prepare 6 tubes of pre-amplification mix

 
 
     Component      Amount (µl)      Final concentration      Mastermix (    samples)        Blue buffer (10×)      0.25      1x               dNTP mix (10mM)      0.1      0.4mM               Preamp Oligo (10 µM)      1      4 µM               Klenow exo- (50 U/µl)      0.5       10 U/µl                H2O      0.65                      Total volume      2.5                 
 
SECTION: Pre-amplification
74. 
Heat the plate to 95 °Cfor 45 sand transfer it to an aluminum rack pre-cooled on ice

SECTION: Pre-amplification
75. 
Centrifuge the plate at 500g for 10 s at 15-25°C
SECTION: Pre-amplification
76. 
Add 2.5 µL of the pre-amplification mix
SECTION: Pre-amplification
77. 
Repeat steps 72-76 five more times
SECTION: Pre-amplification
78. 
Incubate as follows: 
 
     Segment      Temp (ºC)      Duration (min)      Ramp speed (ºC/min)        1      4      5      -        2      4-37      8.25      4        3      37      90      -        4      4      Hold           
 
 

SECTION: Exonuclease I treatment
79. 
Dilute the samples to a volume of98 µLwith nuclease-free water


SECTION: Exonuclease I treatment
80. 
Add 2 µL exonuclease I (20U/µl) to the pre-amplified product and incubate 60 min at 37 °C with the heated lid set to 50 °C

SECTION: Purification
81. 
Add 75 µL AMPure XP beads (0.75:1 ratio) and mix thoroughly by pipetting up and down

 

SECTION: Purification
82. 
Incubate 10 min Room temperature  
In the meantime, prepare Adaptor 2 mix
     Component      Amount   (µl)      Final   concentration      Mastermix   (     samples)        Blue   buffer (10×)      5      1x               dNTP   mix (10mM)      2      0.4mM               Adapter   2 Oligo (10 µM)        2      0.4µM               H2O      40                      Total   volume      49                 
 

SECTION: Purification
83. 
Place on the magnet for 3 minand discard the supernatant

SECTION: Purification
84. 
Add 200 µL of 80% (vol/vol) ethanol while keeping the plate on the magnet then discard ethanol after ±10sec 


SECTION: Purification
85. 
Repeat 84 once. Dry the AMPure XP beads for 5 min Room temperature 

SECTION: Adapter 2 tagging
86. 
Resuspend the beads in 46.5 µL Adapter 2 mix
SECTION: Adapter 2 tagging
87. 
Incubate for 10 min Room temperature 

SECTION: Adapter 2 tagging
88. 
Transfer samples to a new plate 
SECTION: Adapter 2 tagging
89. 
Heat mixture to 95 °Cfor 45 s then immediately cool on ice using an aluminum rack

SECTION: Adapter 2 tagging
90. 
Spin down at 500g for 10 s at 15–25°C
SECTION: Adapter 2 tagging
91. 
Add 1 µL Klenow exo- (50 U/µl), vortex gently, and spin down at 500g for 10 s at 15–25°C 
SECTION: Adapter 2 tagging
92. 
incubate as follows:
 
     Segment      Temp (ºC)      Duration (min)      Ramp speed (ºC/min)        1      4      5      -        2      4-37      8.25      4        3      37      90      -        4      4      Hold           
 
SECTION: Adapter 2 tagging
93. 
In the meantime, prepare 1 tube of  Adapter 2 mix
 
     Component      Amount (µl)      Final concentration      Mastermix (    samples)        Blue buffer (10×)      0.25      1x               dNTP mix (10mM)      0.1      0.4mM               Preamp Oligo (10 µM)      1      4 µM               Klenow exo- (50 U/µl)      0.5       10 U/µl                H2O      0.65                      Total volume      2.5                 
 
SECTION: Adapter 2 tagging
94. 
Heat the plate to 95 °C for  45 s and transfer it to an aluminum rack pre-cooled on ice

SECTION: Adapter 2 tagging
95. 
Centrifuge the plate at 500g for 10 s at 15-25°C

SECTION: Adapter 2 tagging
96. 
Add 2.5 µL of the Adapter 2 mix

SECTION: Adapter 2 tagging
97. 
Incubate as follows: 
 
     Segment      Temp (ºC)      Duration (min)      Ramp speed (ºC/min)        1      4      5      -        2      4-37      8.25      4        3      37      90      -        4      4      Hold           
 
SECTION: Purification
98. 
Add 37.5 µL AMPure XP beads (0.75:1 ratio)

SECTION: Purification
99. 
Incubate 10 min at room temperature
In the meantime, prepare the library amplification mix
 
     Component      Amount (µl)      Final Concentration      Mastermix (    samples)        KAPA HIFI HotStart ReadyMix (2x)      25      1x               PE1.0 (10µM)      1      0.2µM               Nuclease-free water      23                      Total volume      49                 
 
SECTION: Purification
100. 
Place on a magnet for 3 min

SECTION: Purification
101. 
Place on a magnet for 3 minand discard the supernatant

SECTION: Purification
102. 
Add 200 µL ethanol (70%) without disturbing the beads. After 10 s remove ethanol

SECTION: Purification
103. 
Repeat step 102 once then dry beads 5 min at room temperature

SECTION: Library amplification
104. 
Resuspend the beads in 49 µL library amplification mix

SECTION: Library amplification
105. 
Incubate the mixture 10 min Room temperature 

SECTION: Library amplification
106. 
Place on a magnet and transfer supernatant to a new plate
SECTION: Library amplification
107. 
Add 1 µL 10µM reverse iPCRTag primer(containing a sample-specific index)
Amplify as follows:  
     Cycles      Temperature (°C)      Time        1      95      3 min          98      80 s        65      30 s        72      30 s        1      72      3 min        1      4      Hold   
 
 

SECTION: Purification of amplified libraries
108. 
Add 37.5µl AMPure XP beads (0.75:1 ratio) and mix well
SECTION: Purification of amplified libraries
109. Incubate 10 min Room temperature 

SECTION: Purification of amplified libraries
110. 
Place on the magnet for 3 min and discard supernatant
SECTION: Purification of amplified libraries
111. 
Add 200 µL ethanol (70%) without removing the plate from the magnet then discard the ethanol
SECTION: Purification of amplified libraries
112. 
Repeat step 111 once then dry beads 5 min 
SECTION: Purification of amplified libraries
113. 
Resuspend the beads in 15 µL EB 
SECTION: Purification of amplified libraries
115. 
Place on a magnet then transfer supernatant to a new plate
 

SECTION: Purification of amplified libraries
114. 
Incubate 10 min Room temperature 

SECTION: Physical separation of mRNA and gDNA
22.1. 
Manually pipette 10 µL of prepared oligo-dT beads to each well of the sample plate using a multichannel pipette

SECTION: Physical separation of mRNA and gDNA
22.2. 
Mix at maximum speed for 20 min

SECTION: Physical separation of mRNA and gDNA
22.3. 
Place on magnet for 5 min. Aspirate 17.5 µL and transfer to the empty low-bind plate for gDNA collection

SECTION: Physical separation of mRNA and gDNA
22.4. 
Add 15 µL of G&T-seq wash buffer off magnet.

SECTION: Physical separation of mRNA and gDNA
22.5. 
Mix at maximum speed for 10 min

SECTION: Physical separation of mRNA and gDNA
22.6. 
Place on magnet  for 2 min. Aspirate 15 µL and transfer to the empty low-bind plate for gDNA collection

SECTION: Physical separation of mRNA and gDNA
22.7. 
Repeat steps 22.3-22.6 twice more