TITLE: Multiplexed scNOMe-seq protocol based on isolated single nuclei
AUTHORS: Sebastian Pott, Michael Wasney, Nadia Khan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">What follows is the protocol for performing single-cell Nucleosome Occupancy and Methylome sequencing on single nuclei (scNOMe-seq). This plate-based method, which can be used to simultaneously assess nucleosome occupancy and DNA methylation in single nuclei, draws upon the approach laid out in Luo et al., 2018. Everything from the initial nuclei sorting step to the final library preparation that directly precedes sequencing is included in this protocol. This protocol is optimized for non-neuronal nuclei and can be applied to a range of different cell types (e.g., intestinal organoid, fibroblasts).</div><div class = "text-block">This is the method we are using and it is working.</div><div class = "text-block"><span>Adapted from "Robust single-cell DNA methylome profiling with snmC-seq2," by C. Luo et al., 2018, </span><span style = "font-style:italic;">Nature Communications 9</span><span>(1), pp. 1-6. Copyright 2018 by the authors.</span></div></div>

[STEPS]
?. [Nuclei Isolation and GpC Methylation]
Before commencing with nuclei isolation and GpC Methyltransferase step, prepare 384- or 96-well collection plates with digestion mix. This can be prepared the day before and kept at 4ºC.  ABCD1ReagentReaction concentration (based on reaction volume)Volumes for 2 384-well plates (+ 15%) (µL)Volumes for single reaction (µL)2M-Digestion Buffer (2X)1X883.213Proteinase K (1.9%)0.095%88.320.14Distilled H2O794.880.95Total1766.42
ABCD1ReagentReaction concentration (based on reaction volume)Volumes for 2 384-well plates (+ 15%) (µL)Volumes for single reaction (µL)2M-Digestion Buffer (2X)1X883.213Proteinase K (1.9%)0.095%88.320.14Distilled H2O794.880.95Total1766.42
?. [Nuclei Isolation and GpC Methylation]
To prepare Proteinase K, add 1.04 mL of Proteinase K Storage Buffer to one tube of Proteinase K (as per Zymo Kit instructions).
?. [Nuclei Isolation and GpC Methylation]
Also prepare a large quanity of RSB buffer (10X) before beginning nuclei isolation and treatment. From that, make a 1:10 dilution (i.e., RSB buffer 1X) for use in the experiment.
?. [Nuclei Isolation and GpC Methylation]
RSB buffer (10X) recipe:100mM Tris-HCl, pH 7.4100mM NaCl30mM MgCl2This stock can be stored for up to one year at 4ºC.
?. [Nuclei Isolation and GpC Methylation]
Mix enough RSB buffer (1X) for the rest of the nuclei isolation and GpC methylation step (make 5 mL to be safe).
?. [Nuclei Isolation and GpC Methylation]
Start with a suspension of single cells. Count cells and use ~5-10M cells for this protocol.
?. [Nuclei Isolation and GpC Methylation]
Transfer cells to 15 mL Falcon tube.
?. [Nuclei Isolation and GpC Methylation]
Spin for 5 minutes at 500xg at 4°C.
Centrifuge: 500 34, 5 min, 4 10
?. [Nuclei Isolation and GpC Methylation]
Discard supernatant and wash once with ice cold PBS. Spin for 5 minutes at 500xg at 4°C.
Centrifuge: 500 34, 5 min, 4 10
?. [Nuclei Isolation and GpC Methylation]
Discard supernatant and resuspend cells in 1 mL ice-cold RSB buffer (1X).
?. [Nuclei Isolation and GpC Methylation]
Incubate for 10 minutes at room temperature.
[at room temperature]
?. [Nuclei Isolation and GpC Methylation]
Add 15 µL 1% NP-40 solution (0.015% final concentration) to the cell suspension.
[NP-40 (1%)]
The NP-40 concentration might need to be adjusted depending on cell type
?. [Nuclei Isolation and GpC Methylation]
Transfer cell suspension to a dounce tissue grinder (2 mL volume) and burst the cells with 15 strokes of the pestle (both A and B work).
?. [Nuclei Isolation and GpC Methylation]
Transfer lysed cells to a 1.5 mL eppendorf tube.
?. [Nuclei Isolation and GpC Methylation]
Centrifuge cells for 5 minutes at 800xg at 4°C.
Centrifuge: 800 34, 5 min, 4 10
?. [Nuclei Isolation and GpC Methylation]
Discard the supernatant without disturbing the pellet. Wash with 1 mL 1X RSB buffer (without NP-40).
[1X RSB (without NP-40)]
?. [Nuclei Isolation and GpC Methylation]
Incubate in RSB buffer for 30 seconds – 1 minute -
?. [Nuclei Isolation and GpC Methylation]
Centrifuge for 5 minutes at 800xg at 4°C.
Centrifuge: 800 34, 5 min, 4 10
?. [Nuclei Isolation and GpC Methylation]
Discard supernatant and resuspend in 1X GpC methyltransferase buffer such that there are 1M cells per 75 µL.
If there are
?. [Nuclei Isolation and GpC Methylation]
Prepare a 1.5 mL eppendorf tube with GpC Methylase Reaction Mix for incubation:  ABC1ReagentReaction concentration (based on reaction volume)Amount (µL)2GpC methyltransferase buffer (10X) (NEB)0.5X7.53SAM (32mM) (NEB)320µM1.54GpC Methytransferase (4U/µL) (NEB)1.33U/µL505Distilled H2O166Nuclei757total150After adding 75 µL of nuclei to the final mixtures of the above ingredients, pipette to mix.
ABC1ReagentReaction concentration (based on reaction volume)Amount (µL)2GpC methyltransferase buffer (10X) (NEB)0.5X7.53SAM (32mM) (NEB)320µM1.54GpC Methytransferase (4U/µL) (NEB)1.33U/µL505Distilled H2O166Nuclei757total150
?. [Nuclei Isolation and GpC Methylation]
Incubate at 37ºC for 7.5 minutes.
[7.5 minutes]
?. [Nuclei Isolation and GpC Methylation]
Add a boost of 25 µL GpC Methyltransferase (100U) and 0.75 µL 32mM SAM to the nuclei.
[GpC methyltransferase]
[32mM SAM]
?. [Nuclei Isolation and GpC Methylation]
Incubate at 37°C for 7.5 minutes.
[7.5 minutes]
?. [Nuclei Isolation and GpC Methylation]
Add 500 µL 1X PBS and spin for 5 minutes at 800 g at 4°C.
[of 1X PBS]
Centrifuge: 800 34, 5 min, 4 10
?. [Nuclei Isolation and GpC Methylation]
Remove supernatant and resuspend in 0.5-1 mL 1X PBS. –
500 µl
[1X PBS]
?. [Nuclei Isolation and GpC Methylation]
Add 2 drops of Hoechst.
If the cells are resuspended in
?. [Nuclei Isolation and GpC Methylation]
Keep on ice for ~15 minutes before FACS sorting.
[~15 minutes]
?. [Bisulfite Conversion]
Add 15 µL CT conversion reagent to each well of 2 384-well plate. Pipette up and down 8 times to mix the sample.
[CT conversion reagent]
?. [Bisulfite Conversion]
To prepare CT Conversion Reagent add the following buffers to one bottle of CT Conversion Reagent:7.9 mL M-Solubilization Buffer3 mL M-Dilution BufferOnce the reagent is fully dissolved through shaking and vortexing vigorously, add:1.6 mL M-Reaction Buffer Once the reagent is fully dissolved through shaking and vortexing vigorously, add:
?. [Bisulfite Conversion]
Seal the plates with adhesive film and quick spin for 10 seconds at 2,000xg at room temperature.
Centrifuge: 2000 34, 10 s
?. [Bisulfite Conversion]
Place the plate in a thermocycler and run the following program: 98°C    8 minutes 64°C    3.5hrs 4°C      Hold
?. [Bisulfite Conversion]
Prior to cleaning up bisulfite conversion reactions, make Random Primer Solution for each of the 8 primers being used such that each is at a final molarity of 500nM. Keep on ice.  ABCD1ReagentReaction concentration (based on reaction volume)Volumes for 2 384-well plates (+ 8 extra reactions) (µL)Volumes for single reaction (µL)2Random primer stock (100µM)500nM3.640.0353M-Elution Buffer72874Total731.647
ABCD1ReagentReaction concentration (based on reaction volume)Volumes for 2 384-well plates (+ 8 extra reactions) (µL)Volumes for single reaction (µL)2Random primer stock (100µM)500nM3.640.0353M-Elution Buffer72874Total731.647
?. [Bisulfite Conversion]
Place two Zymo-Spin 384-Well DNA Binding Plate on two 2.0 mL 96-Well Deep Well Plates.
?. [Bisulfite Conversion]
Load 80 µL M-Binding Buffer to each well of the Zymo-Spin 384-Well DNA Binding Plates.
[M-Binding Buffer]
?. [Bisulfite Conversion]
Transfer bisulfite conversion reactions to the Zymo-Spin 384-Well DNA Binding Plates. Pipette up and down 8 times to mix the sample.
?. [Bisulfite Conversion]
Centrifuge for 5 minutes at 5,000xg.
Centrifuge: 5000 34, 5 min
?. [Bisulfite Conversion]
Discard the flow through by decanting and add 100 µL M-Wash Buffer to each well of the 384-Well DNA Binding Plates.
[of M-Wash buffer]
?. [Bisulfite Conversion]
Centrifuge for 5 minutes at 5,000xg.
Centrifuge: 5000 34, 5 min
?. [Bisulfite Conversion]
Discard the flow through by decanting and add 50 µL M-Desulphonation Buffer to each well of the 384-Well DNA Binding Plates.
[M-Desulphonation Buffer]
?. [Bisulfite Conversion]
Incubate at room temperature for 15 minutes.
[15 minutes]
?. [Bisulfite Conversion]
Centrifuge for 5 minutes at 5,000xg.
Centrifuge: 5000 34, 5 min
?. [Bisulfite Conversion]
Discard the flow through by decanting and add 100 µL M-Wash Buffer to each well of the 384-Well DNA Binding Plates.
[M-Wash Buffer]
?. [Bisulfite Conversion]
Centrifuge for 5 minutes at 5,000xg.
Centrifuge: 5000 34, 5 min
?. [Bisulfite Conversion]
Repeat wash steps (39 and 40) once more.
?. [Bisulfite Conversion]
Place the 384-Well DNA Binding Plates on 2 new 384-well PCR plates. Add 7 µL Random Primer Solution to each well of the 384-Well DNA Binding Plates (fig. 1).
[Random Primer Solution]
?. [Bisulfite Conversion]
Incubate for 5 minutes at room temperature.
[5 minutes]
?. [Bisulfite Conversion]
Centrifuge for 5 minutes at 5,000xg and discard the 384-Well DNA Binding Plate.
Centrifuge: 5000 34, 5 min
?. [Bisulfite Conversion]
Seal the 384-well PCR plate with adhesive film and store at -20°C for up to 1 week.
[for up to 1 week]
?. [Random-primed DNA synthesis]
Prior to denaturing the samples, make Random Priming Master Mix and keep sealed on ice:  ABCD1ReagentReaction concentration (based on reaction volume)Volumes for 2 384-well plates (+ 20%) (µL)Volumes for single reaction (µL)2Blue Buffer (10X)1X92213Klenow exo (50U/µL)1.25U/µL2310.254dNTP (10mM each)500uM each4610.55Distilled H2O29953.256Total46095
ABCD1ReagentReaction concentration (based on reaction volume)Volumes for 2 384-well plates (+ 20%) (µL)Volumes for single reaction (µL)2Blue Buffer (10X)1X92213Klenow exo (50U/µL)1.25U/µL2310.254dNTP (10mM each)500uM each4610.55Distilled H2O29953.256Total46095
Reaction volume is 10 µL  because it's assumed that 2 µL is lost during the centrifugation in step 44 (Luo et al., 2018)
?. [Random-primed DNA synthesis]
Denature the samples by placing the 384-well PCR plates on a thermocycler and run the following program:95°C    3 minutes
?. [Random-primed DNA synthesis]
Immediately place the plate on ice for 2 minutes.
[2 minutes]
?. [Random-primed DNA synthesis]
Add 5 µL Random Priming Master Mix to each well of the 384-well PCR plates.
[Random Priming Master Mix]
?. [Random-primed DNA synthesis]
Vortex to mix the samples and quick spin for 10 seconds at 2,000xg.
Centrifuge: 2000 34, 10 s
?. [Random-primed DNA synthesis]
Place the plates in a thermocycler and run the following program:4°C      5 minutes 25°C    5 minutes 37°C    60 minutes4°C      Hold
?. [Inactivation of Free Primers & dNTPs]
Add 1.5 µL Exo/rSAP Master Mix to each well of the 384-well PCR plates.   ABCD1ReagentReaction concentration (based on reaction volume)Volumes for 2 384-well plates (+20%) (µL)Volumes for single reaction (µL)2Exonuclease I (20U/µL)1.74U/µL92213rSAP (1U/µL)0.043U/µL4610.54Total13831.5
[Exo/rSAP Mix]
ABCD1ReagentReaction concentration (based on reaction volume)Volumes for 2 384-well plates (+20%) (µL)Volumes for single reaction (µL)2Exonuclease I (20U/µL)1.74U/µL92213rSAP (1U/µL)0.043U/µL4610.54Total13831.5
?. [Inactivation of Free Primers & dNTPs]
Vortex and quick spin for 10 seconds at 2,000xg.
Centrifuge: 2000 34, 10 s
?. [Inactivation of Free Primers & dNTPs]
Place the plate in a thermocycler and run the following program: 37°C    30 min 4°C       Hold
?. [Sample clean-up]
Before proceeding with sample clean-up, prepare enough SPRI beads (14 mL) for the whole experiment. Store beads at 4°C and always take out 30 minutes prior to use.
?. [Sample clean-up]
Mix Sera-Mag SpeedBeads and transfer 280 µL to a 1.5 mL tube.
[Sera-Mag SpeedBeads]
?. [Sample clean-up]
Place beads on a magnetic stand until the solution is clear of beads. Carefully remove the supernatant.
?. [Sample clean-up]
Wash twice with 1 mL TE. For each wash, remove the tube from the magnet and mix by inversion.
[TE]
?. [Sample clean-up]
Resuspend washed beads in 280 µL TE.
[TE]
?. [Sample clean-up]
Add 2.52 g PEG 8000 to a new 50 mL sterile conical tube.
[PEG 8000]
?. [Sample clean-up]
Add 2.8 mL of 5M NaCl to the 50 mL tube.
[5M NaCl]
?. [Sample clean-up]
Add 140 µL 1M Tris-HCl pH=8.0 and 28 µL of 0.5M EDTA pH=8.0 to the 50 mL tube.
[1M Tris-HCl pH=8.0]
[0.5M EDTA pH=8.0]
?. [Sample clean-up]
Add 7-8 mL distilled H2O and mix until PEG 8000 dissolves into solution. –
[distilled H2O]
[distilled H2O]
?. [Sample clean-up]
Add the washed Sera-Mag SpeedBeads to the 50 mL conical tube.
?. [Sample clean-up]
Add enough distilled H2O to bring the total volume up to 14 mL. Vortex before each use.
?. [Sample clean-up]
Add 73.6 µL (0.8x) SPRI beads to each well of a clean 96-well PCR plate.
[SPRI beads]
?. [Sample clean-up]
Pool the samples from the two 384-well PCR plates to one 96-well PCR plate such that each well of the 96-well PCR plate holds a pool of 8 samples, with each of those samples having been indexed with a different distinct random primer during step 42.(two rows of each 384-well plate combine in one row of the 96-well plate.)
?. [Sample clean-up]
Vortex and incubate for 5 minutes at room temperature.
[for 5 minutes]
?. [Sample clean-up]
Quick spin for 10 seconds at 2,000xg at room temperature.
Centrifuge: 2000 34, 10 s
?. [Sample clean-up]
Place the 96-well PCR plate on the DynaMagTM-96 Side Magnet and let stand until the solution in each well is clear of beads (~5 minutes).
?. [Sample clean-up]
Wash beads 3 times with 150 µL fresh 80% EtOH.
[80% EtOH]
?. [Sample clean-up]
Remove all EtOH and let beads dry at room temperature. Do not overdry the beads.
?. [Sample clean-up]
Remove the plate from the magnet and add 10 µL Elution Buffer (Qiagen) to each well. Resuspend beads by pipette.
[Elution Buffer (Qiagen)]
?. [Sample clean-up]
Vortex and incubate for 5 minutes at room temperature.
[for 5 minutes]
?. [Sample clean-up]
Quick spin for 10 seconds at 2,000xg at room temperature.
Centrifuge: 2000 34, 10 s
?. [Sample clean-up]
Place back on magnet and let stand until solution is clear (~5 minutes).
?. [Sample clean-up]
Transfer 10 µL of the supernatant from each well to a clean 96-well PCR plate. The plate can be stored at -20ºC.
[of supernatant]
?. [Adaptase Reaction]
Prior to denaturing the samples, make Adaptase Master Mix and keep sealed on ice:ReagentVolumes for 1 96-well plate (+10 extra reactions) (µL)Volumes for single reaction (µL)Elution Buffer (Qiagen)450.54.25Buffer G12122Reagent G22122Reagent G3132.51.25Enzyme G4530.5Enzyme G5530.5Total111310.5
ReagentVolumes for 1 96-well plate (+10 extra reactions) (µL)Volumes for single reaction (µL)Elution Buffer (Qiagen)450.54.25Buffer G12122Reagent G22122Reagent G3132.51.25Enzyme G4530.5Enzyme G5530.5Total111310.5
?. [Adaptase Reaction]
Immediately place the plate on ice for 2 minutes.
[2 minutes]
?. [Adaptase Reaction]
Place the plate in a thermocycler and run the following program:37°C    30 min 95°C    2 min 4°C      Hold
?. [Library Amplification]
Add 5 µL PCR Primer Mix to every well.
[PCR Primer Mix]
?. [Library Amplification]
Dilute each P5L primer such that the final concentration is 600nM (0.6µM) after the two primers corresponding to each well are combined  ABC1ReagentMix concentration (based on reaction volume)Volume (µL)2P5L stock (100µM)1.2µM1.23Distilled H2O98.84Total100
ABC1ReagentMix concentration (based on reaction volume)Volume (µL)2P5L stock (100µM)1.2µM1.23Distilled H2O98.84Total100
?. [Library Amplification]
Dilute each P7L primer such that the final concentration is 1µM after the two primers corresponding to each well are combined.  ABC1ReagentMix concentration (based on reaction volume)Volume (µL)2P7L stock (100µM)2µM23Distilled H2O984Total100
ABC1ReagentMix concentration (based on reaction volume)Volume (µL)2P7L stock (100µM)2µM23Distilled H2O984Total100
?. [Library Amplification]
To a new 96-well PCR plate, add 3 µL of each P5L primer to individual columns and 3 µL of each P7L primer to individual rows (one P5L primer per column, one P7L primer per row). Each well contains the PCR Primer Mix for the corresponding well in the sample plate. (Fig. 2)
?. [Library Amplification]
Transfer 5 µL of the appropriate primer mix to the wells of the sample plate.
[PCR Primer Mix]
?. [Library Amplification]
Add 25 µL 2X KAPA HiFi Mix to each well.
[2X KAPA HiFi Mix]
?. [Library Amplification]
Vortex and quick spin for 10 seconds at 2,000xg.
Centrifuge: 2000 34, 10 s
?. [Library Amplification]
Place the plate in a thermocycler and run the following program:a. 95°C        2 minutes b. 98°C        30 secondsc. 98°C        15 seconds d. 64°C        30 seconds e. 72°C        2 minutes    Return to step c 14 times (15 cycles total)f. 72°C        5 minutes g. 4°C          Hold
Number of cycles can be adjusted
?. [Library clean-up]
Add 40 µL (0.8x) SPRI Beads to each well of the 96-well PCR plate containing the sample.
[SPRI beads]
?. [Library clean-up]
Vortex and incubate for 5 minutes at room temperature.
[for 5 minutes]
?. [Library clean-up]
Quick spin for 10 seconds at 2,000xg at room temperature.
Centrifuge: 2000 34, 10 s
?. [Library clean-up]
Place 96-well plates on DynaMag™-96 Side Magnet, let stand until solution in wells is clear of beads (~5 minutes).
?. [Library clean-up]
Remove supernatant and wash beads 2 times with 150 µL freshly made 80% EtOH.
[80% EtOH]
?. [Library clean-up]
Remove all EtOH after the last wash and remove plate from magnet. Let beads dry at room temperature. DO NOT overdry beads.
?. [Library clean-up]
Add 25 µL Elution Buffer (Qiagen) and resuspend beads by pipette.
[Elution Buffer (Qiagen)]
?. [Library clean-up]
Vortex and incubate for 5 minutes at room temperature.
[for 5 minutes]
?. [Library clean-up]
Quick spin for 10 seconds at 2,000xg at room temperature.
Centrifuge: 2000 34, 10 s
?. [Library clean-up]
Place back on magnet and let stand until solution is clear of beads (~5 minutes).
?. [Library clean-up]
Combine 25 µL eluent from all wells in each column of the 96-well plate (8 wells per column, 12 columns) into 12 1.5 mL Eppendorf tubes.
[eluent]
?. [Library clean-up]
Add 160 µL (0.8x) SPRI Beads to each 1.5 mL Eppendorf tube. Pipette to mix and incubate for 5 minutes at room temperature.
[SPRI Beads]
?. [Library clean-up]
Place 1.5 mL tubes on DynaMag™-2 Magnet, let stand until solution in tubes in clear of beads (~5 minutes).
?. [Library clean-up]
Remove supernatant and wash beads 2 times with 500 µL fresh 80% EtOH.
[80% EtOH]
?. [Library clean-up]
After the last wash, remove all EtOH and let beads dry at room temperature. DO NOT overdry beads.
?. [Library clean-up]
Add 40 µL Elution Buffer (Qiagen) and resuspend beads by pipet. Incubate for 5 minutes at room temperature.
[Elution Buffer (Qiagen)]
?. [Library clean-up]
Place tubes back on magnet and let stand until solution is clear of beads (~5 minutes).
?. [Library clean-up]
Transfer 40 µL supernatant to 12 new 1.5 mL Eppendorf tubes.
[eluent]
?. [Library clean-up]
Measure concentration of each 1.5 mL Eppendorf tube with Qubit dsDNA BR Assay Kit.
?. [Nuclei Isolation and GpC Methylation]
After FACS, heat plates to 50ºC for 20 minutes in a thermocycler to facilitate nuclei lysis. Store plates at -20ºC or move on to the next step.
[20 minutes]
?. [Adaptase Reaction]
Denature the samples by placing 96-well plates on a thermocycler and run the following program:95°C    3 min
?. [Adaptase Reaction]
Add 10.5 µL Adaptase Master Mix to each well of the 96-well PCR plate.
[Adaptase Master Mix]
?. [Adaptase Reaction]
Vortex and quick spin for 10 seconds at 2,000xg.
Centrifuge: 2000 34, 10 s