Prepare plates containing 2.5µl GpC methylase reaction mixture in each well:

A	B	C	D
Component (initial)	Component(final)	Volume (µl)	Mastermix (110 samples)
M.CviPI reaction buffer (10x)	1x	0.25	27.5
M.CviPI (4U/µl)	2U	0.5	55
SAM (320µM)	160µM	1.25	137.5
IGEPAL (10%)	0.1%	0.025	2.75
RNasein (20U/µl)	1U/µl	0.125	13.75
Nuclease-free water		0.35	38.5

Isolate cells manually or using FACS in 2.5µLof GpC methylase reaction buffer

After cell isolation, spin down plates at ≥1000g for ≥ 10s 4°C

Incubate the samples at 37°C for 0h 15m 0s

Stop reaction by adding 5µL RLT plus buffer

Stoe the plates at-80°C until processed

Add55µLDynabeads into a new Eppendorf tube. Place the tube on a magnet for 0h 2m 0s and discard supernatant

Resuspend beads in 200µL Dynabead solution A ( NaOH, NaCl) 0.1Molarity (M) NaOH, 0.05Molarity (M) NaCl). Place the tube on a magnet for 0h 2m 0s and discard supernatant

Repeat step 8 once

Resuspend beads in 200µL Dynabead solution B ( NaCl) 0.1Molarity (M) NaCl). Place on a magnet for 0h 2m 0s and discard supernatant

Resuspend the beads in55µLof 2x B&W ( NaCl, Tris-HCl, EDTA) 2Molarity (M) NaCl, 10millimolar (mM) Tris-HCl, 1millimolar (mM) EDTA) and 55µL Biotinylated Oligo-dT30VN (100micromolar (µM)).

Incubate 0h 20m 0s on a thermomixer while shaking at 2000rpm at Room temperature

In the meantime, prepare the bead resuspension buffer bead resuspension buffer

A	B
Superscript FS buffer (5x)	220µl
Nuclease-free Water	825µl
RNase inhibitor (20U/µl)	55µl

After adding RNase inhibitor, use beads within 30min

In the meantime, prepare 1x B&W buffer by mixing Nuclease-free water with 2x B&W buffer 1x B&W buffer by mixing 440µL Nuclease-free water with 440µL 2x B&W buffer

Place beads on a magnet for0h 2m 0s and discard supernatant

Resuspend the beads in 200µL 1x B&W buffer. Place beads on a magnet for 0h 2m 0s and discard supernatant

Repeat step 13 three more times

Resuspend the beads in the bead resuspension buffer

Thaw the 96-well plate containing the single cell lysates on ice

Add ERCC spike-ins 1µL 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

Take 4 tubes(1069µLper tube ) of G&T wash buffer( Tris–HCl ,  KCl, MgCl2, 0.5% Tween 20 Solution) 50millimolar (mM) Tris–HCl8.3, 75millimolar (mM) KCl, 3millimolar (mM) MgCl2, 0.5% Tween 20 Solution) and add to each tube137.5µL DTT and RNaseIn 25µL RNaseIn

Add G&T-Seqwash buffer 50µL G&T-Seqwash buffer per well to the“G&T-Seq wash plate”

Add Oligo-dT beads 10µL Oligo-dT beads per well to the“bead plate”

Add an empty non-skirted 96 well plate labeled “gDNA collection”

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 RT master mix

A	B	C	D
Component (C_initial)	C_final	Volume(µl)	Mastermix (110 samples)
dNTP (10mM)	1mM	0.5	55
TSO (100µM)	1µM	0.05	5.5
MgCl2 (1M)	6mM	0.03	3.3
Betain (5M)	1M	1	110
S II First strand buffer (5x)	1x	1	110
DTT (100mM)	5mM	0.25	27.5
Nuclease-free water		1.8	198
RNase inh (20U/µl)	0.5U/µl	0.125	13.75
Superscript reverse transcriptase II (200U/µl)	10U/µl	0.25	27.5

Adding enzyme within less than 30 min before running the Reverse Transcription program

Manually pipette 10µL of prepared oligo-dT beads to each well of the sample plate using a multichannel pipette

Mix at maximum speed for 0h 20m 0s

Place on magnet for 0h 5m 0s. Aspirate 17.5µL and transfer to the empty low-bind plate for gDNA collection

Add 15µL of G&T-seq wash buffer off magnet.

Mix at maximum speed for 0h 10m 0s

Place on magnet for 0h 2m 0s. Aspirate 15µL and transfer to the empty low-bind plate for gDNA collection

Repeat steps 22.3-22.6 twice more

Collect the polyA(+) mRNA plate and using the multi-dispenser dispense RT master mix 5µLRT master mix into each well of the bead-containing 96-well plate

Seal the mRNA and gDNA plates and spin.

Store gDNA at -80°C until processed

Incubate the polyA(+) mRNA 96-well plate on a thermomixer C using the program below (approx. duration 1h 45m 0s)

A	B	C	D
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

In the meantime prepare PCR mastermix PCR mastermix

A	B	C
Component	Volume(µl)	Mastermix (110 samples)
KAPA HiFi HotStart ReadyMix (2x)	6.25	687.5
IS PCR primer (10µM)	0.124	13.64
Nuclease-free water	1.13	124.3

Add PCR reaction mastermix 7.5µLPCR reaction mastermix, seal the plate and centrifuge

Resuspend the beads for0h 0m 30s at 2000rpm using the Thermomixer

Perform cDNA amplification as follows

A	B	C
Cycles	Temperature(°C)	Time
1	98	3 min
18-25	98	20 s
	67	15 s
	72	6 min
1	72	5 min
1	4	Hold

Amplification cycles differ

Add Agencourt AMPure beads 12.5µL Agencourt AMPure beads (1:1 ratio), mix thoroughly by pipetting up and down

Incubate0h 5m 0s at Room temperature

Pellet the beads on a Low-elution magnet for 0h 5m 0s

Remove the supernatant without disturbing the beads

Wash the beads twice with 150µL of freshly prepared 80% ethanol for 0h 0m 10s

Allow the beads to dry for approximately 0h 5m 0s . Resuspend in25µL nuclease-free water. Incubate for 0h 2m 0s Room temperature

Return the 96-well plate to the magnet and allow the Agencourt AMPure beads to settle for 0h 5m 0s

Carefully transfer the supernatant to a new 96-well plate

Dilute the cDNA of each sample to 0.2ng/μl with nuclease-free water

Add 2.5µL Tagment DNA(TD) buffer to a new Hard-Shell skirted 96-well plate

Add diluted cDNA 1.25µL diluted cDNA and amplicon tagment mix 1.25µL amplicon tagment mix (ATM) to TD buffer and mix

Centrifuge the plate at 280x g,20°C

Incubate on a thermal cycler

A	B	C
Segment	Temp(°C)	Duration(min)
1	55	10
2	10	Hold

Add 1.25µLNeutralize Tagment Buffer (NT)

Vortex & spin down at 800x g,20°C

Incubate at Room temperature 0h 5m 0s

Add Index (i7) adapter 1.25µL Index (i7) adapter to each column and Index 2 (i5) adapter 1.25µL Index 2 (i5) adapter to each row

Add3.75µL Nextera PCR mastermix and mix

Centrifuge the samples at 280x g,20°C and amplify as follows:

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

Centrifuge the plate at 280x g,20°C

Purify libraries at a 0.66:1 ratio and elute in EB buffer 12.5µL EB buffer

Pool libraries and quantify using qPCR

Prepare the CT conversion reagent by mixing M-Solubilisation buffer 7.9mL M-Solubilisation buffer and M-Dilution buffer 3mLM-Dilution buffer and 0h 15m 0s vortexing at Room temperature

Finally,add1.6mL M-Reaction buffer and vortex0h 4m 0s at Room temperature

Add AMPure XP beads 32.5µL AMPure XP beads to the gDNA plate (0.65:1 ratio)

Incubate0h 20m 0s Room temperature

Place the plate on the magnet for 0h 20m 0s and discard the supernatant

Wash the beads twice with 200µL80% ethanol

Resuspend the beads in10µL elution buffer, optionally containing 60fg unmethylated lambda DNA

Add 65µL CT conversion reagent without mixing

Incubate the mixture as follows:

A	B	C
Segment	Temperature(ºC)	Duration(min)
1	98	8
2	65	180
3	4	Hold

Mix 300µL M-binding buffer and 5µL MagBinding beads

Transfer the samples to the M-binding buffer - MagBinding beads mix and incubate 0h 5m 0s Room temperature

Pellet the beads on a magnet for 0h 3m 0sand discard the supernatant

Resuspend the beads in200µL M-Wash buffer

Pellet beads on the magnet and discard the supernatant. Resuspend the beads in 100µLM-Desulphonation buffer and incubate 0h 15m 0s``Room temperature

Pellet beads on the magnet and discard the supernatant. Wash the beads twice with 200µL M-Wash buffer

Dry the beads on a heating element at 55°C for 0h 10m 0s

In the meantime, prepare the pre-amplification mix as follows pre-amplification mix as follows

A	B	C	D
Component	Amount (µl)	Final concentration	Mastermix (110 samples)
Blue buffer (10×)	4	1x	440
dNTP mix (10mM)	1.6	0.4mM	176
Preamp Oligo (10 µM)	1.6	O.4 µM	176
H2O	32.8		3608
Total volume	40		4400

Resuspend the beads in a 40µL pre-amplification mix

Incubate the mixture at 55°C for 0h 4m 0s and place it on the magnet

After the beads are pelleted transfer 39µL to a new plate

Incubate the samples 0h 3m 0s at65°C and immediately cool on a pre-cooled aluminum rack

Centrifuge the plate at 500x g

Add 1µL klenow exo- polymerase (50U/µl)

Vortex the samples and amplify as follows:

A	B	C	D
Segment	Temp (ºC)	Duration (min)	Ramp speed (ºC/min)
1	4	5	-
2	4-37	8.25	4
3	37	30	-
4	4	Hold

In the meantime, prepare 6 tubes of pre-amplification mix pre-amplification mix

A	B	C	D
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

Heat the plate to 95°Cfor 0h 0m 45sand transfer it to an aluminum rack pre-cooled on ice

Centrifuge the plate at 500g for 0h 0m 10s at 15-25°C

Add 2.5µL of the pre-amplification mix

Repeat steps 72-76 five more times

Incubate as follows:

A	B	C	D
Segment	Temp (ºC)	Duration (min)	Ramp speed (ºC/min)
1	4	5	-
2	4-37	8.25	4
3	37	90	-
4	4	Hold

Dilute the samples to a volume of98µLwith nuclease-free water

Add exonuclease I 2µL exonuclease I (20U/µl) to the pre-amplified product and incubate 1h 0m 0s at 37°C with the heated lid set to 50°C

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

Incubate 0h 10m 0s Room temperature

In the meantime, prepare Adaptor 2 mix Adaptor 2 mix

A	B	C	D
Component	Amount (µl)	Final concentration	Mastermix (     samples)
Blue buffer (10×)	4.7	1x
dNTP mix (10mM)	1.9	0.4mM
Adapter 2 Oligo (10 µM)	1.9	0.4µM
H2O	38
Total volume	46.5

Place on the magnet for 0h 3m 0sand discard the supernatant

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

Repeat 84 once. Dry the AMPure XP beads for 0h 5m 0s Room temperature

Resuspend the beads in Adapter 2 mix 46.5µL Adapter 2 mix

Incubate for 0h 10m 0s Room temperature

Transfer samples to a new plate

Heat mixture to 95°Cfor 0h 0m 45s then immediately cool on ice using an aluminum rack

Spin down at 500g for 0h 0m 10s at 15–25°C

Add Klenow exo- 1µL Klenow exo- (50 U/µl), vortex gently, and spin down at 500g for 0h 0m 10s at 15–25°C

incubate as follows:

A	B	C	D
Segment	Temp (ºC)	Duration (min)	Ramp speed (ºC/min)
1	4	5	-
2	4-37	8.25	4
3	37	30	-
4	4	Hold

In the meantime, prepare 1 tube of Adapter 2 mix Adapter 2 mix

A	B	C	D
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

Heat the plate to 95°C for 0h 0m 45s and transfer it to an aluminum rack pre-cooled on ice

Centrifuge the plate at 500g for 0h 0m 10s at 15-25°C

Add 2.5µL of the Adapter 2 mix

Incubate as follows:

A	B	C	D
Segment	Temp (ºC)	Duration (min)	Ramp speed (ºC/min)
1	4	5	-
2	4-37	8.25	4
3	37	90	-
4	4	Hold

Add 37.5µL AMPure XP beads ( 0.75:1 ratio )

Incubate 0h 10m 0s at room temperature

In the meantime, prepare the library amplification mix library amplification mix

A	B	C	D
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

Place on a magnet for 0h 3m 0s

Place on a magnet for 0h 3m 0sand discard the supernatant

Add 200µL ethanol (70%) without disturbing the beads. After 0h 0m 10s remove ethanol

Repeat step 102 once then dry beads 0h 5m 0s at room temperature

Resuspend the beads in 49µL library amplification mix

Incubate the mixture 0h 10m 0s Room temperature

Place on a magnet and transfer supernatant to a new plate

Add 1µL 10µM reverse iPCRTag primer (containing a sample-specific index)

Amplify as follows:

A	B	C
Cycles	Temperature (°C)	Time
1	95	3 min
17-20	98	80 s
	65	30 s
	72	30 s
1	72	3 min
1	4	Hold

Add 37.5µl AMPure XP beads (0.75:1 ratio) and mix well

Incubate 0h 10m 0s Room temperature

Place on the magnet for 0h 3m 0s and discard supernatant

Add 200µL ethanol (70%) without removing the plate from the magnet then discard the ethanol

Repeat step 111 once then dry beads 0h 5m 0s

Resuspend the beads in 15µL EB

Incubate 0h 10m 0s Room temperature

Place on a magnet then transfer supernatant to a new plate