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

text stringlengths 0 4.18k
the heterogeneity of Dna methylation within a population of cells necessitates Dna methylome profiling at single-cell resolution.
recently, we developed a single-cell reduced-representation bisulfite sequencing (scrr Bs) technique in which we modified
the original rr Bs method by integrating all the experimental steps before pcr amplification into a single-tube reaction. these
modifications enable scrr Bs to provide digitized methylation information on ~1 million cpG sites within an individual diploid
mouse or human cell at single-base resolution. compared with the single-cell bisulfite sequencing (scBs) technique, scrr Bs covers
fewer cpG sites, but it provides better coverage for cpG islands (cGIs), which are likely to be the most informative elements for
Dna methylation. the entire procedure takes ~3 weeks, and it requires strong molecular biology skills.


Intro Duct Ion
DNA methylation of cy tosine is a well-know n epigenetic modi- but it stil l requires three pur ifications after the end repair/dA
fication that is involved in gene expression regulation1. In mam- tailing , the adapter ligation and the bisulfite conversion. In the
mals, DNA methylation is relatively stable in differentiated cells; scRRBS method, we integ r ated al l the five steps into a sing le-
by cont r ast, g lobal demethy lation and remethy lation occur tub e reac t ion so that DNA pur ificat ion do es not o ccur unt il
dur ing early embr yonic development and pr imordial germ cell the completion of the bisulfite conversion17 (Fig. 1). To achie ve
development2–17. Whole-genome bisulfite sequencing prov ides this, the buffer system and the reaction volumes were modified
a comprehensive v iew of the DNA methylome, but it is ver y to preser ve the different enzy me ac tiv ities at each stage of the
expensive ow ing to deep sequencing of the entire genome18–25. one-tube reaction.
An alternative and cost-efficient technique is RRBS26–28, in which When star ting w ith a single mouse diploid cell, scRRBS covers,
genomic DNA is first digested w ith a restr iction endonuclease on average, 1 million CpG dinucleotides; this accounts for ~40%
(usually MspI) and then size-selected to enr ich for CpG-dense of the CpG sites that can be recovered by standard RRBS using
regions. RRBS prov ides comprehensive DNA methylation infor- thousands of cells (on average, 2.5 million CpG dinucleotides).
mation about the genome. By sequencing ~10% of the mouse or Impor tantly, ~70% of CGIs in the mouse genome can be cap-
human genome, RRBS can reproducibly cover a large propor tion tured. The coverage increases w ith the number of star ting cells,
of the informative CpG sites in the genome, including >70% of and it reaches a plateau at five cells that corresponds to 60% of
promoters, >80% of CGIs and a large number of CGI shores, the coverage achieved using standard RRBS, suggesting that the
enhancers, exons, 3′ untranslated regions (UTRs) and repetitive single-tube approach to some extent affects the coverage that can
elements27,29. However, conventional RRBS techniques require be achieved from the standard RRBS.
nanogram amounts of genomic DNA as star ting material27,30,31,
and they are not applicable to sing le cells. Over v iew of the procedure
The scRRBS approach star ts w ith cell picking and lysis in 5 µl
Development of the procedure of lysis buffer, which contains protease to release the genomic
We recently de veloped scRRBS for profiling DNA methylation DNA. The next three steps, including the MspI digestion, the
at the sing le-cel l le vel10,17. In a t y pical mammalian cel l, there end repair/dA tailing, and the adapter ligation, are accomplished
are only two copies of DNA molecules, and each comes from by adding the corresponding reaction components sequentially,
one of the two parents. If aiming to profile the DNA methy-and the enzy mes in the preceding reactions are inactivated by
lome of a sing le cel l, it is cr ucial to avoid DNA loss as far as heating; Tango buffer is used for all the reactions. The ligation
possible, as al l DNA fr ag ments contain ir replaceable infor ma- is performed by overnig ht incubation using the premethylated
tion. DNA loss is inev itable during purification steps before PCR sequencing adapters and highly concentrated T4 DNA ligase. The
amplification, and there are five such steps in the standard RRBS ligated DNA fragments are directly processed until bisulfite con-
method: (i) genomic DNA pur ification; (ii) restr iction enzy me version, and only after this step is the DNA purified in the pres-
digestion; (iii) end repair and dA tailing (end repair/dA tailing); ence of carr ier tRNA. Next, the DNA is PCR-amplified and the
(iv) adapter ligation; and (v) bisulfite conversion. A more recent fragments between 200 and 700 bp are gel-selected and purified
gel-free RRBS protocol reduces the number of purification steps, as the final librar y for sequencing.

nature protocols \| VOL.10 NO.5 \| 2015 \| 645

## Page 2

protocol

dynamics10. In addition, application of the technique to mouse
embr yonic cells using as few as 20 cells faithfully captured the
CCGG CCGG Mspl digestion DNA methylation status of impr inted genomic reg ions at a
GGCC GGCC (Steps 10–12)
Picking a single cell methylation level of 50% (ref. 34).
(Steps 1–9)

End-repair/dA-tailing Limitations of single-cell methylation profiling techniques
adapter ligation The first limitation of the scRRBS technique stems from the design
(Steps 13–19)
of the RRBS method; that is, although it captures a large por tion
of CGIs and promoters, it prov ides representative, but lower,
U Ucoverage of CpG-sparse reg ions such as enhancers27. It should
Single-tube reaction Bisulfite conversion be noted that the scBS technique is also biased toward CpG-rich
U U (Steps 20–23)
genomic reg ions32, and there are cur rently no sing le-cell DNA
methylation profiling techniques that prov ide whole-genome
PCR amplification coverage. Second, both the scRRBS and the scBS techniques face
(Steps 24–41) the issue of limited overlapping coverage between individual cells.
As mentioned above, the scBS approach suffers more from this
issue. Third, neither approach can discr iminate between DNA
methylation (5mC) and hydroxy methylation (5hmC), and the
A detected methylation information is in fact the sum of 5mC
T High-throughput and 5hmC. Four th, neither technique has been adapted to high-
sequencing
G (Steps 42–44) throug hput platforms such as the microfluidic chip. Fifth, the
C mapping efficiencies of both scRRBS and scBS are relatively low
\| (~25% on average) when star ting w ith a sing le cell. For scRRBS,
Figure 1 Flowchart of the experimental procedures of the scRRBS the mapping efficiency increases w ith the number of star ting
technique. Notably, we integrated cell lysis, MspI digestion, end repair/dA cells, and it reaches a plateau of 50%, which is close to that of
tailing, adapter ligation and bisulfite treatment into a single-tube reaction standard RRBS17.
to avoid unnecessary DNA loss.

Experimental design
Comparison of scRRBS and single-cell bisulfite sequencing Star ting mater ial. We have successfully applied this protocol to a
32 variety of mammalian cells, including human and mouse embr y-
Recently, Smallwood et al. repor ted an alternative scBS tech- onic stem cells (mESCs), oocy tes, sperm, early preimplantation
nique. The authors used the sing le-tube reaction str ateg y to blastomeres and cancer cells. The method is likely to be applica-
improve a prev ious protocol that was based on bisulfite conver- ble to most mouse and human cell t y pes, regardless of whether
sion followed by random pr iming33. When star ting w ith a sin- they are isolated from tissues or cell culture. However, it is rec-
g le mouse diploid cell, scBS covers, on average, 3.7 million CpG ommended to selec t the healthiest-looking cel ls w ith good
dinucleotides. Althoug h the overall coverage of scBS is hig her mor pholog y to avoid p otent ial genomic DNA de g r adat ion
than that of the scRRBS, it has two limits. First, althoug h scBS before cell lysis. We have not yet tested this method on any non-
includes more CpG-sparse regions, its coverage of CGI is lower mammalian cells. Both sing le cells and a small number of cells
than scRRBS. Second, scBS profiles the genome in a relatively ran- (several to hundreds of cells, such as cells isolated from a sing le
dom and less consistent manner, meaning that there is less overlap blastocyst) can ser ve as the star ting material for scRRBS. If you
between the individual CpG sites covered in different single cells. are aiming to profile the DNA methylation of a population w ith
Thus, the two approaches prov ide complementar y information, a limited number of cells, it will be more efficient to pool the cells
and the choice of which method to use should depend on the aim for analysis and perform several biological replicates. This analy-

the heterogeneity of Dna methylation within a population of cells necessitates Dna methylome profiling at single-cell resolution.

recently, we developed a single-cell reduced-representation bisulfite sequencing (scrr Bs) technique in which we modified

the original rr Bs method by integrating all the experimental steps before pcr amplification into a single-tube reaction. these

modifications enable scrr Bs to provide digitized methylation information on ~1 million cpG sites within an individual diploid

mouse or human cell at single-base resolution. compared with the single-cell bisulfite sequencing (scBs) technique, scrr Bs covers

fewer cpG sites, but it provides better coverage for cpG islands (cGIs), which are likely to be the most informative elements for

Dna methylation. the entire procedure takes ~3 weeks, and it requires strong molecular biology skills.

Intro Duct Ion

DNA methylation of cy tosine is a well-know n epigenetic modi- but it stil l requires three pur ifications after the end repair/dA

fication that is involved in gene expression regulation1. In mam- tailing , the adapter ligation and the bisulfite conversion. In the

mals, DNA methylation is relatively stable in differentiated cells; scRRBS method, we integ r ated al l the five steps into a sing le-

by cont r ast, g lobal demethy lation and remethy lation occur tub e reac t ion so that DNA pur ificat ion do es not o ccur unt il

dur ing early embr yonic development and pr imordial germ cell the completion of the bisulfite conversion17 (Fig. 1). To achie ve

development2–17. Whole-genome bisulfite sequencing prov ides this, the buffer system and the reaction volumes were modified

a comprehensive v iew of the DNA methylome, but it is ver y to preser ve the different enzy me ac tiv ities at each stage of the

expensive ow ing to deep sequencing of the entire genome18–25. one-tube reaction.

An alternative and cost-efficient technique is RRBS26–28, in which When star ting w ith a single mouse diploid cell, scRRBS covers,

genomic DNA is first digested w ith a restr iction endonuclease on average, 1 million CpG dinucleotides; this accounts for ~40%

(usually MspI) and then size-selected to enr ich for CpG-dense of the CpG sites that can be recovered by standard RRBS using

regions. RRBS prov ides comprehensive DNA methylation infor- thousands of cells (on average, 2.5 million CpG dinucleotides).

mation about the genome. By sequencing ~10% of the mouse or Impor tantly, ~70% of CGIs in the mouse genome can be cap-

human genome, RRBS can reproducibly cover a large propor tion tured. The coverage increases w ith the number of star ting cells,

of the informative CpG sites in the genome, including >70% of and it reaches a plateau at five cells that corresponds to 60% of

promoters, >80% of CGIs and a large number of CGI shores, the coverage achieved using standard RRBS, suggesting that the

enhancers, exons, 3′ untranslated regions (UTRs) and repetitive single-tube approach to some extent affects the coverage that can

elements27,29. However, conventional RRBS techniques require be achieved from the standard RRBS.

nanogram amounts of genomic DNA as star ting material27,30,31,

and they are not applicable to sing le cells. Over v iew of the procedure

The scRRBS approach star ts w ith cell picking and lysis in 5 µl

Development of the procedure of lysis buffer, which contains protease to release the genomic

We recently de veloped scRRBS for profiling DNA methylation DNA. The next three steps, including the MspI digestion, the

at the sing le-cel l le vel10,17. In a t y pical mammalian cel l, there end repair/dA tailing, and the adapter ligation, are accomplished

are only two copies of DNA molecules, and each comes from by adding the corresponding reaction components sequentially,

one of the two parents. If aiming to profile the DNA methy-and the enzy mes in the preceding reactions are inactivated by

lome of a sing le cel l, it is cr ucial to avoid DNA loss as far as heating; Tango buffer is used for all the reactions. The ligation

possible, as al l DNA fr ag ments contain ir replaceable infor ma- is performed by overnig ht incubation using the premethylated

tion. DNA loss is inev itable during purification steps before PCR sequencing adapters and highly concentrated T4 DNA ligase. The

amplification, and there are five such steps in the standard RRBS ligated DNA fragments are directly processed until bisulfite con-

method: (i) genomic DNA pur ification; (ii) restr iction enzy me version, and only after this step is the DNA purified in the pres-

digestion; (iii) end repair and dA tailing (end repair/dA tailing); ence of carr ier tRNA. Next, the DNA is PCR-amplified and the

(iv) adapter ligation; and (v) bisulfite conversion. A more recent fragments between 200 and 700 bp are gel-selected and purified

gel-free RRBS protocol reduces the number of purification steps, as the final librar y for sequencing.

nature protocols | VOL.10 NO.5 | 2015 | 645

## Page 2

protocol

dynamics10. In addition, application of the technique to mouse

embr yonic cells using as few as 20 cells faithfully captured the

CCGG CCGG Mspl digestion DNA methylation status of impr inted genomic reg ions at a

GGCC GGCC (Steps 10–12)

Picking a single cell methylation level of 50% (ref. 34).

(Steps 1–9)

End-repair/dA-tailing Limitations of single-cell methylation profiling techniques

adapter ligation The first limitation of the scRRBS technique stems from the design

(Steps 13–19)

of the RRBS method; that is, although it captures a large por tion

of CGIs and promoters, it prov ides representative, but lower,

U Ucoverage of CpG-sparse reg ions such as enhancers27. It should

Single-tube reaction Bisulfite conversion be noted that the scBS technique is also biased toward CpG-rich

U U (Steps 20–23)

genomic reg ions32, and there are cur rently no sing le-cell DNA

methylation profiling techniques that prov ide whole-genome

PCR amplification coverage. Second, both the scRRBS and the scBS techniques face

(Steps 24–41) the issue of limited overlapping coverage between individual cells.

As mentioned above, the scBS approach suffers more from this

issue. Third, neither approach can discr iminate between DNA

methylation (5mC) and hydroxy methylation (5hmC), and the

A detected methylation information is in fact the sum of 5mC

T High-throughput and 5hmC. Four th, neither technique has been adapted to high-

sequencing

G (Steps 42–44) throug hput platforms such as the microfluidic chip. Fifth, the

C mapping efficiencies of both scRRBS and scBS are relatively low

| (~25% on average) when star ting w ith a sing le cell. For scRRBS,

Figure 1 Flowchart of the experimental procedures of the scRRBS the mapping efficiency increases w ith the number of star ting

technique. Notably, we integrated cell lysis, MspI digestion, end repair/dA cells, and it reaches a plateau of 50%, which is close to that of

tailing, adapter ligation and bisulfite treatment into a single-tube reaction standard RRBS17.

to avoid unnecessary DNA loss.

Experimental design

Comparison of scRRBS and single-cell bisulfite sequencing Star ting mater ial. We have successfully applied this protocol to a

32 variety of mammalian cells, including human and mouse embr y-

Recently, Smallwood et al. repor ted an alternative scBS tech- onic stem cells (mESCs), oocy tes, sperm, early preimplantation

nique. The authors used the sing le-tube reaction str ateg y to blastomeres and cancer cells. The method is likely to be applica-

improve a prev ious protocol that was based on bisulfite conver- ble to most mouse and human cell t y pes, regardless of whether

sion followed by random pr iming33. When star ting w ith a sin- they are isolated from tissues or cell culture. However, it is rec-

g le mouse diploid cell, scBS covers, on average, 3.7 million CpG ommended to selec t the healthiest-looking cel ls w ith good

dinucleotides. Althoug h the overall coverage of scBS is hig her mor pholog y to avoid p otent ial genomic DNA de g r adat ion

than that of the scRRBS, it has two limits. First, althoug h scBS before cell lysis. We have not yet tested this method on any non-

includes more CpG-sparse regions, its coverage of CGI is lower mammalian cells. Both sing le cells and a small number of cells

than scRRBS. Second, scBS profiles the genome in a relatively ran- (several to hundreds of cells, such as cells isolated from a sing le

dom and less consistent manner, meaning that there is less overlap blastocyst) can ser ve as the star ting material for scRRBS. If you

between the individual CpG sites covered in different single cells. are aiming to profile the DNA methylation of a population w ith

Thus, the two approaches prov ide complementar y information, a limited number of cells, it will be more efficient to pool the cells

and the choice of which method to use should depend on the aim for analysis and perform several biological replicates. This analy-