TITLE: scNMT-seq
AUTHORS: Stephen Clark

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Here we describe the full wetlab protocol for scNMT-seq (single-cell nucleosome position, methylome and transcriptome sequencing), a plate-based single-cell multi-omic method.</div><div class = "text-block">In scNMT-seq, a GpC methyltransferase is used to label non-nucleosome bound DNA thus encoding chromatin accessiblity information within the genomic DNA. Bisulfite conversion is then used to discriminate the two chromatin states which can be read from GpC dinucleotide positions. Endogenous DNA methylation is read from the CpG positions of the same molecules and gene expression data is obtained by capturing poly-A RNA on magnetic beads and pysically separating before the gDNA is bisulfite converted. This mRNA on beads is pocessed using Smart-seq2. The protocol is carried out in 96w plates and typically takes 3-4 days to complete.</div></div>

[STEPS]
?. [Single-cell collection and methylase reaction]
Collect single cells in Centrifuge at ≥1000g for ≥10s.
[  of freshly prepared GpC methylase reaction mix (keep chilled on ice)]
We have successfully used both flow sorting and manual pipetting for single-cell collections.
?. [Single-cell collection and methylase reaction]
Incubate on a thermocycler:
37 °C
?. [Single-cell collection and methylase reaction]
Add Centrifuge at ≥1000g for ≥10s.
[RLT plus]
?. [Single-cell collection and methylase reaction]
Store at
-80 °C
?. [Prepare oligo-dT  on beads]
Pipette into a microcentrifuge tube.  Place on a magnet and remove supernatent.
[ Dynabeads (MyOne Streptavidin C1) ]
?. [Prepare oligo-dT  on beads]
Resuspend beads in  by pippetting. Place on a magnet and remove supernatent.
[solution A  (0.1M NaOH, 0.05M NaCl)]
?. [Prepare oligo-dT  on beads]
Resuspend beads in . Place on magnet and remove supernatent.
[solution B (0.1M NaCl)]
?. [Prepare oligo-dT  on beads]
Resuspend beads in . Place on magnet and remove supernatent.
[2x B&W (2 M NaCl, 10 mM Tris-HCl, 1 mM EDTA)]
?. [Prepare oligo-dT  on beads]
Resuspend beads in   and .
[2x B&W]
[bio-Smartseq2-dT (100uM)]
bio-Smartseq2-dT: 5'-Biotin-TEG-AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3' (IDT, purified with HPLC and resuspended in nuclease free water)
?. [Prepare oligo-dT  on beads]
Rotate tube containing beads such that beads remain in solution for a minimum of .
Beads can be prepared in advance and stored at   for up to 1 month
?. [Wash oligo dT beads and prepare for dispensing]
Place on magnet and remove supernatent. Resuspend beads in . Place on magnet and remove supernatent.
[1x B&W buffer (1M NaCl, 5mM Tris, 0.5mM EDTA)]
?. [Wash oligo dT beads and prepare for dispensing]
Place on magnet and remove supernatent. Resuspend beads in: ,,,
[Nuclease free water]
[5x first strand buffer (Invitrogen, Superscript II)]
[RNasin (Promega)]
[Diluted ERCC spike-in ]
This assumes 2.5ul GpC reaction plus 5ul RLTplus. If using bigger volumes, adjust upward in proportion.
ERCC spike-in is optional. The concentration used will depend on cell type (RNA content).
?. [mRNA separation]
Manually pipette of prepared oligo-dT beads to each well of the sample plate using a multichannel pipette.
10 µl
?. [mRNA separation]
Prepare G&T-seq wash buffer:  AB1Reagent96 well plate2Nuclease free water343035x First strand buffer (Invitrogen Superscript II)4304DTT (100mM, Invitrogen Superscript II)4305Tween-20 (100%)226Recombinant RNasin (40U/ul)437Total4355
AB1Reagent96 well plate2Nuclease free water343035x First strand buffer (Invitrogen Superscript II)4304DTT (100mM, Invitrogen Superscript II)4305Tween-20 (100%)226Recombinant RNasin (40U/ul)437Total4355
?. [mRNA separation]
Prepare RT mastermix:  ABC1Reagentamount per sample96 well plate (x110)2Nuclease free water3.715408.6535x First strand buffer (Superscript II)22204Betaine (5M)22205DTT (100mM, Superscript II)0.5556dNTPs (10mM each)11107MgCl2 (1M)0.066.68Smartseq2 TSO (100uM)0.1119Recombinant RNasin (40U/ul)0.12513.7510Superscript II (200U/ul)0.55511Total101100
ABC1Reagentamount per sample96 well plate (x110)2Nuclease free water3.715408.6535x First strand buffer (Superscript II)22204Betaine (5M)22205DTT (100mM, Superscript II)0.5556dNTPs (10mM each)11107MgCl2 (1M)0.066.68Smartseq2 TSO (100uM)0.1119Recombinant RNasin (40U/ul)0.12513.7510Superscript II (200U/ul)0.55511Total101100
?. [mRNA separation]
Mix at maximum speed for
We perform mRNA separation steps on an Agilent Bravo liquid handling robot. If performing this step manually, intermittent vortexing for 10 minutes achieves the same result.
?. [mRNA separation]
Place on magnet until beads pelette. Aspirate  and transfer to empty lobind plate for gDNA collection.
17.5 µl
?. [mRNA separation]
Add of G&T-seq wash buffer off magnet.
15 µl
?. [mRNA separation]
Mix at maximum speed for
If performing manually, a thorough vortexing here suffices.
?. [mRNA separation]
[Repeat steps 22-25 twice more.]
Lysate (17.5ul) combined with 3 washes (15ul each) should now have been collected into the gDNA plate
Pause point. gDNA can be frozen at -20C for later processing.
?. [mRNA separation]
Add  to beads.
[RT mastermix]
?. [mRNA separation]
Mix at maximum speed for
?. [Reverse transcription]
Incubate on a thermocycler as follows:
42 °C
50 °C
60 °C
Vortex plate at 30 and 60 minute time points to resuspend beads.
?. [cDNA amplification]
Add  PCR mastermix by hand using a multichannel pipette.  ABC1Reagentamount per cell96 well plate2KAPA Hifi 2x Readymix1112103ISPCR primer (10uM)0.2527.54Water0.7582.55Total121320
12 µl
ABC1Reagentamount per cell96 well plate2KAPA Hifi 2x Readymix1112103ISPCR primer (10uM)0.2527.54Water0.7582.55Total121320
?. [cDNA amplification]
Cycle as follows:18 Cycles of:Followed by:
98 °C
98 °C
67 °C
72 °C
72 °C
[pause - usually overnight]
4 °C
Cycle number depends on cell type. 18 is sufficient for mouse ES cells and embryo cells.
Pause point. cDNA can be frozen at -20C for later processing.
?. [cDNA purification using 1x AMPure XP]
Add  AMPure XP beads. Mix thoroughly and incubate for 5 minutes.
This section is performed outside of pre-PCR room.
18 µl
?. [Genomic DNA purification]
Add  AMPure XP beads to gDNA samples. Vortex thoroughly and incubate for
50 µl
?. [Prepare bisulfite conversion reagent]
Bisulfite reagent is prepared according to manufacturers instructions (Zymo EZ-methylation direct):Add and  to a bottle of CT Conversion Reagent. Mix at room temperature with frequent vortexing or shaking for at least 10 minutes. Add   and vortex thoroughly.
[M-Solubilization Buffer ]
[M-Dilution Buffer]
[M-Reaction Buffer]
?. [Bisulfite conversion]
Place gDNA sample plate on magnet to pellet beads.
?. [Bisulfite conversion]
Add of prepared CT Conversion Reagent solution to each sample on the magnet.
65 µl
Do not mix sample and beads with CT reagent – beads should remain pelleted during conversion to avoid excessive disolving of magnetic beads.
?. [Bisulfite conversion]
Incubate on a thermocycler as follows:
98 °C
64 °C
[pause until purification (maximum duration of overnight)]
?. [Desulphonation and purification]
Add   to , mix and dispense per well of deepwell plate using multichannel and reservoir.
[Zymo Magbinding beads]
[M-Binding buffer]
305 µl
?. [Desulphonation and purification]
Add sample to deepwell plate containing beads and binding buffer. Mix thoroughly then transfer  back to sample plate, mix thoroughly and transfer back to deepwell plate to maximise recovery of bisuflite converted DNA. Mix for
100 µl
?. [Desulphonation and purification]
Prepare first strand master mix for elution:  ABC1Reagentamount per sample96 well plate (x120)2Nuclease free water31.83816310x Blue buffer (Enzymatics)44804dNTP mix (10mM each)1.61925First strand oligo (10uM)1.61926Total394680
ABC1Reagentamount per sample96 well plate (x120)2Nuclease free water31.83816310x Blue buffer (Enzymatics)44804dNTP mix (10mM each)1.61925First strand oligo (10uM)1.61926Total394680
First strand oligo: /5SpC3/CTACACGACGCTCTTCCGATCTNNNNNN (IDT, HPLC purified).This primer contains the Illumina PE read 1 sequence.
?. [Desulphonation and purification]
Resuspend beads in . incubate at
[first strand synthesis mix]
60 °C
?. [Desulphonation and purification]
Place on magnet until beads pellet. Transfer supernatent to fresh lobind PCR plate.
20 µl
?. [First strand synthesis]
Place sample plate on thermocycler at then immediately cool on ice.
65 °C
?. [First strand synthesis]
Add Klenow exo- (50 U/ul, Enzymatics) using multichannel and PCR strip.
1 µl
?. [First strand synthesis]
Incubate on a thermocyler as follows:Slow ramp from  to  at 15s per 1C hold.
4 °C
4 °C
37 °C
37 °C
4 °C
?. [First strand synthesis]
95 °C for 45 seconds then immediately cool 4 °C using two ice blocks.
95 °C
?. [First strand synthesis]
Add   ABC1Reagentamount per sample96 well plate (x110)2Nuclease free water0.6571.5310x Blue buffer0.2527.54dNTP mix (10mM each)0.1115First strand oligo (10uM)11106Klenow exo- (50U/ul)0.5557Total2.5275
[first-strand extra cycles mix]
ABC1Reagentamount per sample96 well plate (x110)2Nuclease free water0.6571.5310x Blue buffer0.2527.54dNTP mix (10mM each)0.1115First strand oligo (10uM)11106Klenow exo- (50U/ul)0.5557Total2.5275
First strand oligo: /5SpC3/CTACACGACGCTCTTCCGATCTNNNNNN (IDT, HPLC purified).This primer contains the Illumina PE read 1 sequence.
?. [First strand synthesis]
[Repeat first strand synthesis an additional 3 times]
?. [Exonuclease treatment]
ABC1Reagentamount per sample96 well plate(x120)2Nuclease free water4857603Exonuclease I (NEB)22404Total50600
[Exonuclease mix]
ABC1Reagentamount per sample96 well plate(x120)2Nuclease free water4857603Exonuclease I (NEB)22404Total50600
?. [Exonuclease treatment]
37 °C for 1 hour.
37 °C
?. [1st Strand Purification]
Add  per well of a deepwell plate. Transfer  sample to deepwell plate. Mix thoroughly and incubate at room temperature for 10 minutes.
[AMPureXP beads]
100 µl
?. [1st Strand Purification]
Place plate on magent and wait until beads pellet. Remove supernatent.
?. [1st Strand Purification]
Add  off the magnet and mix thoroughly with pipetting.
[80% ethanol]
?. [1st Strand Purification]
Place plate on magent and wait until beads pellet. Remove supernatent.
?. [1st Strand Purification]
Resuspend beads in   ABC1Reagentamount per sample96 well plate (x120)2Nuclease free water404800310x Blue buffer56004dNTP mix (10mM each)22405Second strand oligo (10uM)22406Total495880
[second strand master mix]
ABC1Reagentamount per sample96 well plate (x120)2Nuclease free water404800310x Blue buffer56004dNTP mix (10mM each)22405Second strand oligo (10uM)22406Total495880
Second strand oligo: TGCTGAACCGCTCTTCCGATCTNNNNNN (HPLC purified from IDT).This primer contains the Illumina PE read 2 sequence. Users wishing to use Truseq indexing primers (instead of iPCRTag) should redesign this so that it matches the Truseq read 2 sequence (TCAGACGTGTGCTCTTCCGATC).
?. [1st Strand Purification]
Transfer  second strand mix containing beads to a fresh 96w PCR plate.
?. [Second strand synthesis]
Incubate on a thermocyler:, then immediatly cool on ice.
98 °C
?. [Second strand synthesis]
Add  and incubate:Slow ramp from  to  at 15s per 1C hold.
[Klenow exo- (50U/ul, Enzymatics)]
4 °C
4 °C
37 °C
37 °C
4 °C
?. [2nd Strand Purification]
Prepare an aliquot of AMPure buffer by pelleting AMPure XP beads using a magnet or centrifuge and taking the supernatent.
?. [2nd Strand Purification]
Add and per well of a deepwell plate.
[AMPure buffer]
[water]
?. [2nd Strand Purification]
Transfer sample, containing beads, to the deepwell plate containing AMPure buffer and water. Mix thoroughly then incubate at room temperature
?. [2nd Strand Purification]
Place plate on magnet and wait until beads pellet.
?. [2nd Strand Purification]
Remove supernatent.
?. [2nd Strand Purification]
Wash twice with 80% ethanol.
?. [2nd Strand Purification]
Resuspend beads in :  ABC1Reagentamount per sample96 well plate(x120)2Water2226403KAPA Hifi readymix (2x)2530004PE1.0 (10uM)11205Total485760
[PCR master mix]
ABC1Reagentamount per sample96 well plate(x120)2Water2226403KAPA Hifi readymix (2x)2530004PE1.0 (10uM)11205Total485760
PE1.0: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T (IDT, HPLC)This primer contains the full Illumina P5 and PE read 1 sequences.
?. [2nd Strand Purification]
Add
[iTAG indexing primer (5uM)]
We prepare iTAG primers in 96w plates for this step.iPCRTag: CAAGCAGAAGACGGCATACGAGATXXXXXXXXGAGATCGGTCTCGGCATT CCTGCTGAACCGCTCTTCCGATC*T (IDT, HPLC, XXXXXXXX = 8nt index)This primer contains the full Illumina P7 sequence, followed by an index, followed by the Illumina PE read 2 sequence.
?. [Library amplification]
Cycle as follows:14 cycles of:Followed by: hold.
95 °C
94 °C
65 °C
72 °C
72 °C
4 °C
?. [Library purification]
All subsequent steps are performed outside of pre-PCR room.
?. [Library purification]
Make 2 pools of 48 libraries (5ul each) in 1.5ml tubes.
The number of samples per pool depends on the number of indexes used. e.g. we typically use only 48 indexes and so make two pools of 48 samples each.
?. [Single-cell collection and methylase reaction]
Prepare GpC methylase reaction buffer:  ABC1Reagentamount per sample96 well plate (x120)2Nuclease free water1.4251713GpC methyltransferase buffer (10x)0.25304SAM0.01251.55IGEPAL CA-630 (1%)0.25306RNase-in (40U/ul)0.06257.57M.CviPI (4U/ul)0.5608Total2.5300
ABC1Reagentamount per sample96 well plate (x120)2Nuclease free water1.4251713GpC methyltransferase buffer (10x)0.25304SAM0.01251.55IGEPAL CA-630 (1%)0.25306RNase-in (40U/ul)0.06257.57M.CviPI (4U/ul)0.5608Total2.5300
?. [Prepare oligo-dT  on beads]
[: Repeat solution A wash one more time]
?. [Prepare oligo-dT  on beads]
[: Repeat solution B wash once.]
?. [Prepare oligo-dT  on beads]
?. [Wash oligo dT beads and prepare for dispensing]
?. [mRNA separation]
Place on magnet until beads pelette. Aspirate  and transfer to empty lobind plate for gDNA collection.
15 µl
?. [Genomic DNA purification]
Place on a magnet until beads pellet. Remove supernatent, wash twice with . Remove supernatent. Immediatley resuspend in of nuclease free water.
[80% ethanol]
10 µl
We use an Agilent Bravo for these wash steps.
?. [Desulphonation and purification]
Plate on magnet until beads pellet. Remove supernatent.
?. [Desulphonation and purification]
Add . Mix thoroughly.
[freshly prepared 80% ethanol]
?. [Desulphonation and purification]
[: Repeat once more.]
?. [Desulphonation and purification]
Place on magnet until beads pellet. Remove supernatent.
?. [Desulphonation and purification]
Add . Mix thoroughly.
[M-Desulphonation buffer (Zymo)]
?. [Desulphonation and purification]
Incubate
Incubate for  ≥ 15 minutes and ≤ 25 minutes
?. [Desulphonation and purification]
Place on magnet and remove supernatent.
?. [Desulphonation and purification]
Add . Mix thoroughly.
[freshly prepared 80% ethanol]
?. [Desulphonation and purification]
Place on magnet until beads pellet. Remove supernatent.
?. [Desulphonation and purification]
Dry beads
60 °C
?. [Desulphonation and purification]
[: Repeat elution once more.]
?. [cDNA purification using 1x AMPure XP]
Elute cDNA in
[water]
?. [cDNA purification using 1x AMPure XP]
Check quality of a subet (e.g. 11 samples) of cDNA samples using a Bioanalyzer high sensitivty DNA chip.
Fragment size range of 1kb to 5kb. Concentration of 100 to 500 pg/ul. If concentration is higher then dilute samples with water prior to Nextera XT library prep.
?. [Nextera XT library prepation]
In a 96 well or  a 384 well plate, add 2.25ul Tagmentation mastermix to each well:  ABCD1Reagentamount per sample96 well plate (x120)384 well plate2Tagment DNA buffer1.51806503Amplicon tagment mix0.75903254Total2.25270975
This section is performed outside of pre-PCR room.
ABCD1Reagentamount per sample96 well plate (x120)384 well plate2Tagment DNA buffer1.51806503Amplicon tagment mix0.75903254Total2.25270975
?. [Nextera XT library prepation]
In a thermocycler incubate as follows:,
55 °C
[pause]
?. [Nextera XT library prepation]
Add using multichannel pipette. Centrifuge then vortex to mix and centrifuge again.
[NT buffer]
?. [Nextera XT library prepation]
Add Add Using a multichannel pipette. Centrifuge and vortex to mix.
[NPM (from Nextera XT kit)]
[pre-mixed and diluted i7 and i5 primers]
We prepare pre-mixed indexing primers in 96w plates for this step. Using multichannel pipettes, transfer each of 8 i5 primers to 12 columns and each of 12 i7 primers to 8 rows, then dilute 5-fold with water.
?. [Nextera XT library prepation]
Cycle as follows:10 cycles of:Followed by: hold.
72 °C
95 °C
95 °C
65 °C
72 °C
72 °C
4 °C
?. [Nextera XT library prepation]
Pool of each indexed cDNA library. Purify using 0.7x AMPure XP beads with 2x 80% ethanol washes.
1 µl
?. [Nextera XT library prepation]
QC using Bioanalyzer.
?. [cDNA purification using 1x AMPure XP]
Place on magnet until beads precipitate. Remove supernatent. Wash twice with 80% ethanol. Remove supernatent and air-dry beads for 5 minutes at room temperature.
?. [First strand synthesis]
Incubate on a thermocylcer as follows:Slow ramp from  to  at 15s per 1C
4 °C
4 °C
37 °C
37 °C
?. [First strand synthesis]
For the fifth and final round, incubate for an additional 1 hour: hold.
37 °C
4 °C
?. [Nextera XT library prepation]
Using a multi-channel pipette, add 0.75ul of cDNA sample (100-500pg/ul) to the mastermix. Vortex the plate and spin down.
?. [1st Strand Purification]
Add  on the magnet.
[80% ethanol]
?. [1st Strand Purification]
Remove supernatent and air dry at
50 °C
?. [2nd Strand Purification]
Remove supernatent. Air dry
50 °C
?. [2nd Strand Purification]
Tranfer samples to fresh 96w PCR plate.
?. [Library purification]
Purify each pool with  (i.e. 0.7x ratio), 2x 80% ethanol washes and elute in .
[AMPure XP beads]
[water]
?. [Library purification]
Purify each pool a second time  (i.e. 0.7x ratio), 2x 80% ethanol washes and elute in .
[AMPure XP beads]
[water]
?. [Library purification]
QC using a Bioanalyser high sensitivity chip.
Pooled and purified scBS libraries should have a fragment length of 300-1000bp (average 450-500). Pools containing smaller fragments (especially