sequencing/scg-protocols-v1 · Datasets at Hugging Face

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water to allow it to dissolve for at least 10 min, and then dilute it with resuspension buffer (part of the MethylCode bisulfite conversion kit) and
nuclease-free water to 10 ng/µl as the working concentration; divide the 300 µl of dilution buffer (part of the MethylCode bisulfite conversion kit)
solution into 1.5-ml Eppendorf DNA LoBind tubes and store the aliquots directly to the CT conversion powder (also part of the same kit). Mix the
at −80 °C for no more than 6 months. solution by brief intermittent vortexing until the solution becomes clear.
End-repair dNTP mix Mix and dilute the dATP, dGTP and dCTP stock Always keep the dissolved CT conversion reagent in the dark, and then store
solutions (100 mM each) to 1 mM, 0.1 mM and 0.1 mM, respectively. Divide it for up to 2 weeks at −20 °C. Avoid repeated freeze/thaw cycles.
the solutions into aliquots in 1.5-ml Eppendorf DNA LoBind tubes and store Native polyacr ylamide TBE gel, 12% (w t/vol) Add 7.5 ml of 30% (wt/vol)
them at −20 °C for no more than 6 months. acrylamide, 5 ml of 5× TBE buffer, 180 µl of 10% (wt/vol) AP, 16 µl of
Maintenance of mESCs mESCs are maintained without feeders in the presence TEMED and nuclease-free water up to 25 ml. Mix the solution well by
of LIF in DMEM containing 20% FBS for routine passage without modification. vortexing, pipette it into the gap between the glass gel-casting plates and
Unmethylated λ-DNA spike-in Unmethylated λ-DNA (0.3 µg/µl) should insert the ten-well combs. Let the gel stand at room temperature for at
be diluted with nuclease-free water and quantified by a Qubit fluorometer least 1 h to ensure complete polymerization.  cr ItIcal Gels can be stored
and the Qubit dsDNA HS assay kit to ~60 fg/µl. This spike-in stock solution at 4 °C in sealed packs for up to 2 weeks.





proce Dure
cell culture, single-cell isolation and lysis ● tIMInG 4–5 d
 cr ItIcal Incubations before bisulfite conversion (i.e., before Step 18) should be performed at a temperature no higher
than 80 °C; higher temperatures increase the risk of denaturing the double-stranded genomic DNA.
 cr ItIcal The nuclease-free water and all of the tubes used in this protocol, including the 0.2-ml thin-walled PCR tubes
and the 1.5-ml Eppendorf DNA LoBind tubes, should be UV-sterilized for at least 5 min to prevent contamination.
 cr ItIcal All steps performed with a PCR thermocycler should be carried out on an instrument with a heated lid to avoid
liquid evaporation from the reaction tubes.

1\| Aspirate the medium from mESCs maintained in 60-mm cell culture dishes (at 80% confluency) and wash the entire
surface area with 1× PBS. Aspirate the PBS and add sufficient 0.05% (wt/vol) trypsin to cover the cell growth area.
Return the dishes to a 37 °C incubator and incubate them for 5 min.
 cr ItIcal step One 60-mm dish at 80% confluency provides sufficient mESCs for scRRBS.

2\| After the cells have detached, add 5 ml of DMEM with 20% (vol/vol) FBS to inactivate the trypsin, gently pipette the
suspension to obtain single cells and collect the cell suspension into a 15-ml Falcon conical tube.

3\| Centrifuge the mixture at 300g for 5 min at room temperature and aspirate the supernatant.

4\| Wash the cell pellet with 1 ml of PBS-BSA, centrifuge it at 300g for 5 min at room temperature and aspirate the
supernatant. Repeat this step once more, and finally suspend the pellet in 20–50 µl of PBS-BSA solution.
 cr ItIcal step Always use PBS-BSA instead of PBS in this step to minimize nonspecific binding to the reaction tubes.







nature protocols \| VOL.10 NO.5 \| 2015 \| 649

## Page 6

protocol


5\| Prepare the cell lysis buffer as set out in the following table. Mix it well by vortexing, centrifuge the mixture at 9,000g
for 1 min at 4 °C, and then divide the mixture into 0.2-ml thin-walled PCR tubes (4.8 µl in each tube).

component amount per sample (ml) Final concentration

Tris-EDTA (1 M Tris, 0.1 M EDTA) 0.1 20 mM Tris, 2 mM EDTA

1 M KCl 0.1 20 mM

10% (vol/vol) Triton X-100 0.15 0.3% (vol/vol)

20 mg/ml protease 0.25 1 mg/ml

Nuclease-free water up to 4.8 —

 cr ItIcal step Always include at least one ‘picking-buffer-only’ control when preparing the lysis buffer.
 cr ItIcal step Be sure to use protease instead of proteinase K when preparing lysis buffer, as protease can be
heat-inactivated at a much lower temperature than proteinase K.
 cr ItIcal step If you are starting from single sperm cells, add 10 mM DTT to the lysis buffer to decondense the sperm nucleus.

6\| Carefully transfer single cells from Step 4 into the tubes prepared in Step 5, taking care to avoid forming bubbles.
We have found mouth pipetting to be the most accurate way to achieve this40–42; however, alternative approaches include
fluorescence-activated cell sorting (FACS)43, laser-assisted microdissection44 or microfluidic platforms45,46.
 cr ItIcal step The volume of carry-over PBS-BSA during mouth pipetting of single cells should be no >0.2 µl.
 cr ItIcal step It is essential to include a ‘picking-buffer-only’ (cell lysis buffer without cells) control to evaluate possible
contamination during subsequent procedures.
! caut Ion Be careful with the glass capillary; handle it with appropriate protection.
! caut Ion Not all institutions permit mouth pipetting for manipulation of single cells, especially when working on certain
types of sample (e.g., primary human tissues or some infectious samples). Alternative approaches that comply with
institutional, local and governmental regulations should be adopted.

7\| Centrifuge the tubes at 9,000g for at least 1 min at 4 °C to ensure that the cell is completely seeded into the lysis buffer.
 cr ItIcal step Do not vortex the mixture, as the genomic DNA is easily fragmented.
? trou Bles Hoot InG

8\| Lyse the cells at 50 °C for 3 h, and then incubate them at 75 °C for 30 min to inactivate the protease.
 cr ItIcal step This step should be performed in a PCR thermocycler rather than in a heat block or a water bath because of
its capacity for stable temperature control.

9\| Centrifuge the tubes at 9,000g for 1 min at 4 °C and immediately place the tubes on ice.
? trou Bles Hoot InG
 pause poInt The lysate can be frozen at −80 °C for no longer than 3 months. However, we strongly recommend proceeding
to the next step immediately.

MspI digestion ● tIMInG 3–4 h
10\| Prepare the MspI digestion mixture, as described in the following table. Mix it well by vortexing, and then centrifuge the

water to allow it to dissolve for at least 10 min, and then dilute it with resuspension buffer (part of the MethylCode bisulfite conversion kit) and

nuclease-free water to 10 ng/µl as the working concentration; divide the 300 µl of dilution buffer (part of the MethylCode bisulfite conversion kit)

solution into 1.5-ml Eppendorf DNA LoBind tubes and store the aliquots directly to the CT conversion powder (also part of the same kit). Mix the

at −80 °C for no more than 6 months. solution by brief intermittent vortexing until the solution becomes clear.

End-repair dNTP mix Mix and dilute the dATP, dGTP and dCTP stock Always keep the dissolved CT conversion reagent in the dark, and then store

solutions (100 mM each) to 1 mM, 0.1 mM and 0.1 mM, respectively. Divide it for up to 2 weeks at −20 °C. Avoid repeated freeze/thaw cycles.

the solutions into aliquots in 1.5-ml Eppendorf DNA LoBind tubes and store Native polyacr ylamide TBE gel, 12% (w t/vol) Add 7.5 ml of 30% (wt/vol)

them at −20 °C for no more than 6 months. acrylamide, 5 ml of 5× TBE buffer, 180 µl of 10% (wt/vol) AP, 16 µl of

Maintenance of mESCs mESCs are maintained without feeders in the presence TEMED and nuclease-free water up to 25 ml. Mix the solution well by

of LIF in DMEM containing 20% FBS for routine passage without modification. vortexing, pipette it into the gap between the glass gel-casting plates and

Unmethylated λ-DNA spike-in Unmethylated λ-DNA (0.3 µg/µl) should insert the ten-well combs. Let the gel stand at room temperature for at

be diluted with nuclease-free water and quantified by a Qubit fluorometer least 1 h to ensure complete polymerization.  cr ItIcal Gels can be stored

and the Qubit dsDNA HS assay kit to ~60 fg/µl. This spike-in stock solution at 4 °C in sealed packs for up to 2 weeks.

proce Dure

cell culture, single-cell isolation and lysis ● tIMInG 4–5 d

 cr ItIcal Incubations before bisulfite conversion (i.e., before Step 18) should be performed at a temperature no higher

than 80 °C; higher temperatures increase the risk of denaturing the double-stranded genomic DNA.

 cr ItIcal The nuclease-free water and all of the tubes used in this protocol, including the 0.2-ml thin-walled PCR tubes

and the 1.5-ml Eppendorf DNA LoBind tubes, should be UV-sterilized for at least 5 min to prevent contamination.

 cr ItIcal All steps performed with a PCR thermocycler should be carried out on an instrument with a heated lid to avoid

liquid evaporation from the reaction tubes.

1| Aspirate the medium from mESCs maintained in 60-mm cell culture dishes (at 80% confluency) and wash the entire

surface area with 1× PBS. Aspirate the PBS and add sufficient 0.05% (wt/vol) trypsin to cover the cell growth area.

Return the dishes to a 37 °C incubator and incubate them for 5 min.

 cr ItIcal step One 60-mm dish at 80% confluency provides sufficient mESCs for scRRBS.

2| After the cells have detached, add 5 ml of DMEM with 20% (vol/vol) FBS to inactivate the trypsin, gently pipette the

suspension to obtain single cells and collect the cell suspension into a 15-ml Falcon conical tube.

3| Centrifuge the mixture at 300g for 5 min at room temperature and aspirate the supernatant.

4| Wash the cell pellet with 1 ml of PBS-BSA, centrifuge it at 300g for 5 min at room temperature and aspirate the

supernatant. Repeat this step once more, and finally suspend the pellet in 20–50 µl of PBS-BSA solution.

 cr ItIcal step Always use PBS-BSA instead of PBS in this step to minimize nonspecific binding to the reaction tubes.

nature protocols | VOL.10 NO.5 | 2015 | 649

## Page 6

protocol

5| Prepare the cell lysis buffer as set out in the following table. Mix it well by vortexing, centrifuge the mixture at 9,000g

for 1 min at 4 °C, and then divide the mixture into 0.2-ml thin-walled PCR tubes (4.8 µl in each tube).

component amount per sample (ml) Final concentration

Tris-EDTA (1 M Tris, 0.1 M EDTA) 0.1 20 mM Tris, 2 mM EDTA

1 M KCl 0.1 20 mM

10% (vol/vol) Triton X-100 0.15 0.3% (vol/vol)

20 mg/ml protease 0.25 1 mg/ml

Nuclease-free water up to 4.8 —

 cr ItIcal step Always include at least one ‘picking-buffer-only’ control when preparing the lysis buffer.

 cr ItIcal step Be sure to use protease instead of proteinase K when preparing lysis buffer, as protease can be

heat-inactivated at a much lower temperature than proteinase K.

 cr ItIcal step If you are starting from single sperm cells, add 10 mM DTT to the lysis buffer to decondense the sperm nucleus.

6| Carefully transfer single cells from Step 4 into the tubes prepared in Step 5, taking care to avoid forming bubbles.

We have found mouth pipetting to be the most accurate way to achieve this40–42; however, alternative approaches include

fluorescence-activated cell sorting (FACS)43, laser-assisted microdissection44 or microfluidic platforms45,46.

 cr ItIcal step The volume of carry-over PBS-BSA during mouth pipetting of single cells should be no >0.2 µl.

 cr ItIcal step It is essential to include a ‘picking-buffer-only’ (cell lysis buffer without cells) control to evaluate possible

contamination during subsequent procedures.

! caut Ion Be careful with the glass capillary; handle it with appropriate protection.

! caut Ion Not all institutions permit mouth pipetting for manipulation of single cells, especially when working on certain

types of sample (e.g., primary human tissues or some infectious samples). Alternative approaches that comply with

institutional, local and governmental regulations should be adopted.

7| Centrifuge the tubes at 9,000g for at least 1 min at 4 °C to ensure that the cell is completely seeded into the lysis buffer.

 cr ItIcal step Do not vortex the mixture, as the genomic DNA is easily fragmented.

? trou Bles Hoot InG

8| Lyse the cells at 50 °C for 3 h, and then incubate them at 75 °C for 30 min to inactivate the protease.

 cr ItIcal step This step should be performed in a PCR thermocycler rather than in a heat block or a water bath because of

its capacity for stable temperature control.

9| Centrifuge the tubes at 9,000g for 1 min at 4 °C and immediately place the tubes on ice.

? trou Bles Hoot InG

 pause poInt The lysate can be frozen at −80 °C for no longer than 3 months. However, we strongly recommend proceeding

to the next step immediately.

MspI digestion ● tIMInG 3–4 h

10| Prepare the MspI digestion mixture, as described in the following table. Mix it well by vortexing, and then centrifuge the