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

text stringlengths 0 4.18k
the solutions into aliquots in 1.5-ml Eppendorf DNA LoBind tubes and store Native polyacrylamide TBE gel, 12% (wt/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 ofrights 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
All 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
Inc. be diluted with nuclease-free water and quantified by a Qubit fluorometer least 1 h to ensure complete polymerization.  CRITICAL 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.
America,
Nature PROCEDURE
2015 Cell CRITICALculture, Incubationssingle-cell isolationbefore bisulfiteand lysisconversion● TIMING(i.e.,4–5befored 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.
 CRITICAL 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.
 CRITICAL 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.
 CRITICAL 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.
 CRITICAL 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 —

 CRITICAL STEP Always include at least one ‘picking-buffer-only’ control when preparing the lysis buffer.
 CRITICAL 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.
 CRITICAL 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 includereserved. fluorescence-activated cell sorting (FACS)43, laser-assisted microdissection44 or microfluidic platforms45,46.
rights  CRITICALCRITICAL STEPSTEP TheIt is volumeessentialof tocarry-overinclude aPBS-BSA‘picking-buffer-only’during mouth(cellpipettinglysis bufferof singlewithoutcells cells)shouldcontrolbe no >0.2to evaluateµl. possible
All contamination during subsequent procedures.
! CAUTION Be careful with the glass capillary; handle it with appropriate protection.
Inc. ! CAUTION 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.America,
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.
 CRITICAL STEP Do not vortex the mixture, as the genomic DNA is easily fragmented.Nature ? TROUBLESHOOTING
2015 8\| Lyse the cells at 50 °C for 3 h, and then incubate them at 75 °C for 30 min to inactivate the protease.
©  CRITICAL 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.
? TROUBLESHOOTING
 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
mixture at 9,000g for 1 min at 4 °C.

Component Amount per reaction (ml) Final amount
10 U/µl MspI 0.9 9 U

10× Tango buffer 1.8 1×
Unmethylated λ-DNA (60 fg/µl) 1 60 fg

Nuclease-free water up to 13 —

 CRITICAL STEP Always ensure that the λ spike-in accounts for ~1% (mass/mass) of the total genomic DNA of every picked
single cell. For example, if you are starting with a single diploid mammalian cell, add 1 µl (~60 fg) of unmethylated λ-DNA to
the digestion mixture.


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

## Page 7

protocol

the solutions into aliquots in 1.5-ml Eppendorf DNA LoBind tubes and store Native polyacrylamide TBE gel, 12% (wt/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 ofrights 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

All 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

Inc. be diluted with nuclease-free water and quantified by a Qubit fluorometer least 1 h to ensure complete polymerization.  CRITICAL 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.

America,

Nature PROCEDURE

2015 Cell CRITICALculture, Incubationssingle-cell isolationbefore bisulfiteand lysisconversion● TIMING(i.e.,4–5befored Step 18) should be performed at a temperature no higher

 CRITICAL 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.

 CRITICAL 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.

 CRITICAL 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.

 CRITICAL 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 —

 CRITICAL STEP Always include at least one ‘picking-buffer-only’ control when preparing the lysis buffer.

 CRITICAL 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.

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

rights  CRITICALCRITICAL STEPSTEP TheIt is volumeessentialof tocarry-overinclude aPBS-BSA‘picking-buffer-only’during mouth(cellpipettinglysis bufferof singlewithoutcells cells)shouldcontrolbe no >0.2to evaluateµl. possible

All contamination during subsequent procedures.

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

Inc. ! CAUTION 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.America,

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.

 CRITICAL STEP Do not vortex the mixture, as the genomic DNA is easily fragmented.Nature ? TROUBLESHOOTING

2015 8| Lyse the cells at 50 °C for 3 h, and then incubate them at 75 °C for 30 min to inactivate the protease.

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.

? TROUBLESHOOTING

 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

mixture at 9,000g for 1 min at 4 °C.

Component Amount per reaction (ml) Final amount

10 U/µl MspI 0.9 9 U

10× Tango buffer 1.8 1×

Unmethylated λ-DNA (60 fg/µl) 1 60 fg

Nuclease-free water up to 13 —

 CRITICAL STEP Always ensure that the λ spike-in accounts for ~1% (mass/mass) of the total genomic DNA of every picked

single cell. For example, if you are starting with a single diploid mammalian cell, add 1 µl (~60 fg) of unmethylated λ-DNA to

the digestion mixture.

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

## Page 7

protocol