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

text stringlengths 0 4.18k
dinucleotides. Although the overall coverage of scBS is higher2015 morphology to avoid potential genomic DNA degradation
© than that of the scRRBS, it has two limits. First, although 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 single 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 single
dom and less consistent manner, meaning that there is less overlap blastocyst) can serve as the starting 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 with
Thus, the two approaches provide complementary 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
of specific studies. sis provides information about the average methylation level for
each covered CpG site within the population, which is similar to
Applications of scRRBS conventional RRBS. Alternatively, if you wish to study cell hetero
The advantage of the scRRBS method is its applicability to sub geneity with respect to DNA methylation within a population, or
nanogram levels of DNA as starting material, down to a single if there are only one or a few highly heterogeneous cells within the
cell. This is particularly useful when the starting materials are biological sample, individual cells must be analyzed; in this case,
very limited and precious, such as mammalian early embryos and dozens (10–100) of single cells are required to obtain accurate
primordial germ cells. It also enables the heterogeneity of DNA measurement of DNA methylation for a certain cell type. This
methylomes among individual cells to be studied, which may have analysis detects the methylation status of covered CpG sites in
important roles in biological processes such as cell differentiation, a digitized manner, as each individual mammalian cell contains
memory formation and oncogenesis. As examples, we have suc only two copies of DNA molecules, or one copy of DNA molecule
cessfully applied scRRBS to individual mouse sperm cells, and we when a haploid germ cell is analyzed.
detected each recovered CpG site as either fully unmethylated or
fully methylated, which is expected because sperm cells contain Controls. It is important to avoid any DNA contamination in
only a single copy of the haploid genome17. We have also applied scRRBS experiments. All reagents and consumables used in this
this approach to individual human and mouse pronuclei iso protocol should be free of exogenous DNA, and ‘picking-buffer-
lated from zygotes, and we observed global DNA demethylation only’ controls (i.e., only pick the PBS-BSA buffer but no cells

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

## Page 3

protocol

into the lysis buffer) are always essential for evaluating possible may be a consequence of the low sequence complexity of
contamination during the whole experimental process. scRRBS libraries.

Enzyme choice. The restriction endonuclease MspI (C↓CGG) is Data analysis. Bioinformatics analysis methods for DNA
commonly used in RRBS, and it is the focus of this scRRBS proto methylation profiling data of whole-genome bisulfite sequencing
col. MspI digestion is strongly biased toward CGIs and promoters; and RRBS have been reported previously, which are designed to
however, other restriction endonucleases such as TaqI (T↓CGA) map bisulfite-converted sequence reads to a reference genome
could be chosen to capture different genomic regions. In addi and to determine cytosine methylation states at single-base
tion, digestion using two or more endonucleases may increase resolution10,17. Customized scripts written in Perl or Python
the genomic coverage. can be applied for discarding low-quality reads, trimming
adapter sequences and removing additional bases that are arti
Sequencing. Different sequencing platforms can be used, includ ficially introduced during the end-repair steps. Alternatively,
ing the Illumina HiSeq 2000 and HiSeq 2500 platforms we use. the Trim Galore tool can be used (http://www.bioinformatics.
We generally sequence each scRRBS library to ~10 million babraham.ac.uk/projects/trim_galore/). Bioinformatic tools such
100-bp paired-end reads. Increasing the sequencing depth to as Bismark35, BSMAP36 or BS Seeker37 can be applied for the
20 million reads will only slightly raise the genomic coverage. alignment of the bisulfite-converted sequences and downstream
In our experience, a cluster density of 80% of the regular or analysis such as visualization of the data, comparison between
mixing scRRBS libraries with other non-bisulfite-sequencing samples and identification of differentially methylated regions.
libraries will increase the yield and quality of the reads, which The Bismark program works well in our hands.
reserved.
rights MATERIALS
All REAGENTS • Phusion high-fidelity (HF) PCR master mix with HF buffer (New England
 CRITICAL All reagents used in this experiment must be free of nucleases. BioLabs, cat. no. M0531S)
Inc. ! CAUTION All samples from animals or humans should be processed • Qubit dsDNA high-sensitivity (HS) assay kit (Thermo Scientific, according to the guidelines of the local government or institution. cat. no. Q32854)
• Cell line or single cells of interest. In the PROCEDURE, we use as an example • SYBR Gold nucleic acid gel stain (10,000×; Thermo Scientific,
mESCs maintained in conventional DMEM, containing 20% (vol/vol) FBS cat. no. S-11494) ! CAUTION This reagent is harmful if swallowed or
in the presence of leukemia inhibitory factor (LIF) inhaled, and it causes irritation to the skin, the eyes and the respiratoryAmerica, • Tris-EDTA solution (100×; Sigma-Aldrich, cat. no. T9285-100ML) tract. Handle it using appropriate equipment.
• Triton X-100 (Sigma-Aldrich, cat. no. T8787-50ML) ! CAUTION Triton • Glycerol (Sigma-Aldrich, cat. no. G5516-100ML)
X-100 is harmful if swallowed or inhaled, and it causes irritation to the skin, • QIAquick PCR purification kit (Qiagen, cat. no. 28106)Nature the eyes and the respiratory tract. Handle it using appropriate equipment. • Ethanol (Sigma-Aldrich, cat. no. E7023) ! CAUTION Ethanol is flammable.
 CRITICAL Triton X-100 should be stored at room temperature (20–25 °C) Handle it using appropriate equipment.
for at most 1 year. • Primers for two rounds of PCR amplification (these oligos can be2015 • Potassium chloride solution (KCl; 1 M; Sigma-Aldrich, synthesized and HPLC-purified from IDT): QP...
©
cat. no. 60142-100ML-F) CACCGA-3′ and QP2: 5′-CAAGCAGAAGACGGCATACGA-3′
• Glycerol (Sigma-Aldrich, cat. no. G5516-100ML) • High-sensitivity next-generation sequencing (NGS) fragment analysis kit
• Protease (7.5 Anson units (AU); Qiagen, cat. no. 19155) (Advanced Analytical Technologies, cat. no. DNF-486-0500)
• Nuclease-free water (Ambion, cat. no. AM9932) • 40% Acryl/Bis (29:1; Amresco, cat. no. 0311-1L) ! CAUTION This compound
• MspI (10 U/µl; Thermo Scientific, cat. no. ER0541) is harmful if swallowed or inhaled, and it causes irritation to the skin, the
• Tango buffer (10×; Thermo Scientific, cat. no. BY5) eyes and the respiratory tract. Handle it using appropriate equipment.
• λ-DNA (dam–, dcm–; 0.3 µg/µl, Thermo Scientific, cat. no. SD0021) • TBE buffer (5×; Amresco, cat. no. J885-1L)
• Klenow fragment (exo–, 5 U/µl; Thermo Scientific, cat. no. EP0422) • Ammonium persulfate (AP; Sigma-Aldrich, cat. no. A3678-100G)
• dATP (100 mM; New England BioLabs, cat. no. N0440S) ! CAUTION AP is harmful if swallowed or inhaled, and it causes irritation
• dCTP (100 mM; New England BioLabs, cat. no. N0441S) to the skin, the eyes and the respiratory tract. Handle it using appropriate
• dGTP (100 mM; New England BioLabs, cat. no. N0442S) equipment.
• T4 DNA ligase (highly concentrated; 30 Weiss U/µl; Thermo Scientific, • N,N,N′,N′-tetramethylethylenediamine (TEMED; Sigma-Aldrich,
cat. no. EL0013) cat. no. T9281-100ML) ! CAUTION TEMED is harmful if swallowed or
• ATP (100 mM; Thermo Scientific, cat. no. R0441) inhaled, and it causes irritation to the skin, the eyes and the respiratory
• Adapters, part of the Illumina TruSeq DNA sample preparation kits tract. Handle it using appropriate equipment.
(Illumina, cat. no. FC-121-2001)  CRITICAL Illumina TruSeq DNA • Ammonium acetate (Amresco, cat. no. 0103-500G)
adapters are highly recommended. If they are not available, ensure that the • Magnesium acetate tetrahydrate (Amresco, cat. no. 0131-500G)
cytosines (Cs) of the ordered oligos are methyl group–modified (mCs) and • SDS (Amresco, cat. no. S0227-500G)
double HPLC-purified. The oligo sequences of these indexed adapters are • EDTA (Amresco, cat. no. 0322-1KG)
listed in Table 1. • PBS buffer (pH 7.2; 1×; Gibco, cat. no. 14249-95)
• MethyCode bisulfite conversion kit (50 reactions; Thermo Scientific, • Leukemia inhibitory factor (LIF; 10 million units/1 ml, Millipore,
cat. no. MECOV-50) cat. no. ESG1107)
• tRNA (Roche, cat. no. 10109517001) • DMEM: nutrient mixture F-12 (DMEM/F-12; Gibco, cat. no. 11320-033)
• PfuTurbo Cx hotstart DNA polymerase (2.5 U/µl; Agilent Technologies, • FBS (Gibco, cat. no. 12483-020)
cat. no. 600412) • Trypsin-EDTA, 0.05% (wt/vol) (1×; Gibco, cat. no. 25300-062)
• dNTP mix (10 mM each; Clontech, cat. no. D4030RA) • Albumin, acetylated from bovine serum (Ac-BSA; Sigma-Aldrich,
• Agencourt AMPure XP beads (Beckman Coulter; cat. no. A63881) cat. no. B8894)


nature protocols \| VOL.10 NO.5 \| 2015 \| 647

## Page 4

protocol

Table 1 \| Oligo sequences of adapters.

dinucleotides. Although the overall coverage of scBS is higher2015 morphology to avoid potential genomic DNA degradation

includes more CpG-sparse regions, its coverage of CGI is lower mammalian cells. Both single 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 single

dom and less consistent manner, meaning that there is less overlap blastocyst) can serve as the starting 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 with

Thus, the two approaches provide complementary 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

of specific studies. sis provides information about the average methylation level for

each covered CpG site within the population, which is similar to

Applications of scRRBS conventional RRBS. Alternatively, if you wish to study cell hetero

The advantage of the scRRBS method is its applicability to sub geneity with respect to DNA methylation within a population, or

nanogram levels of DNA as starting material, down to a single if there are only one or a few highly heterogeneous cells within the

cell. This is particularly useful when the starting materials are biological sample, individual cells must be analyzed; in this case,

very limited and precious, such as mammalian early embryos and dozens (10–100) of single cells are required to obtain accurate

primordial germ cells. It also enables the heterogeneity of DNA measurement of DNA methylation for a certain cell type. This

methylomes among individual cells to be studied, which may have analysis detects the methylation status of covered CpG sites in

important roles in biological processes such as cell differentiation, a digitized manner, as each individual mammalian cell contains

memory formation and oncogenesis. As examples, we have suc only two copies of DNA molecules, or one copy of DNA molecule

cessfully applied scRRBS to individual mouse sperm cells, and we when a haploid germ cell is analyzed.

detected each recovered CpG site as either fully unmethylated or

fully methylated, which is expected because sperm cells contain Controls. It is important to avoid any DNA contamination in

only a single copy of the haploid genome17. We have also applied scRRBS experiments. All reagents and consumables used in this

this approach to individual human and mouse pronuclei iso protocol should be free of exogenous DNA, and ‘picking-buffer-

lated from zygotes, and we observed global DNA demethylation only’ controls (i.e., only pick the PBS-BSA buffer but no cells

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

## Page 3

protocol

into the lysis buffer) are always essential for evaluating possible may be a consequence of the low sequence complexity of

contamination during the whole experimental process. scRRBS libraries.

Enzyme choice. The restriction endonuclease MspI (C↓CGG) is Data analysis. Bioinformatics analysis methods for DNA

commonly used in RRBS, and it is the focus of this scRRBS proto methylation profiling data of whole-genome bisulfite sequencing

col. MspI digestion is strongly biased toward CGIs and promoters; and RRBS have been reported previously, which are designed to

however, other restriction endonucleases such as TaqI (T↓CGA) map bisulfite-converted sequence reads to a reference genome

could be chosen to capture different genomic regions. In addi and to determine cytosine methylation states at single-base

tion, digestion using two or more endonucleases may increase resolution10,17. Customized scripts written in Perl or Python

the genomic coverage. can be applied for discarding low-quality reads, trimming

adapter sequences and removing additional bases that are arti

Sequencing. Different sequencing platforms can be used, includ ficially introduced during the end-repair steps. Alternatively,

ing the Illumina HiSeq 2000 and HiSeq 2500 platforms we use. the Trim Galore tool can be used (http://www.bioinformatics.

We generally sequence each scRRBS library to ~10 million babraham.ac.uk/projects/trim_galore/). Bioinformatic tools such

100-bp paired-end reads. Increasing the sequencing depth to as Bismark35, BSMAP36 or BS Seeker37 can be applied for the

20 million reads will only slightly raise the genomic coverage. alignment of the bisulfite-converted sequences and downstream

In our experience, a cluster density of 80% of the regular or analysis such as visualization of the data, comparison between

mixing scRRBS libraries with other non-bisulfite-sequencing samples and identification of differentially methylated regions.

libraries will increase the yield and quality of the reads, which The Bismark program works well in our hands.

reserved.

rights MATERIALS

All REAGENTS • Phusion high-fidelity (HF) PCR master mix with HF buffer (New England

 CRITICAL All reagents used in this experiment must be free of nucleases. BioLabs, cat. no. M0531S)

Inc. ! CAUTION All samples from animals or humans should be processed • Qubit dsDNA high-sensitivity (HS) assay kit (Thermo Scientific, according to the guidelines of the local government or institution. cat. no. Q32854)

• Cell line or single cells of interest. In the PROCEDURE, we use as an example • SYBR Gold nucleic acid gel stain (10,000×; Thermo Scientific,

mESCs maintained in conventional DMEM, containing 20% (vol/vol) FBS cat. no. S-11494) ! CAUTION This reagent is harmful if swallowed or

in the presence of leukemia inhibitory factor (LIF) inhaled, and it causes irritation to the skin, the eyes and the respiratoryAmerica, • Tris-EDTA solution (100×; Sigma-Aldrich, cat. no. T9285-100ML) tract. Handle it using appropriate equipment.

• Triton X-100 (Sigma-Aldrich, cat. no. T8787-50ML) ! CAUTION Triton • Glycerol (Sigma-Aldrich, cat. no. G5516-100ML)

X-100 is harmful if swallowed or inhaled, and it causes irritation to the skin, • QIAquick PCR purification kit (Qiagen, cat. no. 28106)Nature the eyes and the respiratory tract. Handle it using appropriate equipment. • Ethanol (Sigma-Aldrich, cat. no. E7023) ! CAUTION Ethanol is flammable.

 CRITICAL Triton X-100 should be stored at room temperature (20–25 °C) Handle it using appropriate equipment.

for at most 1 year. • Primers for two rounds of PCR amplification (these oligos can be2015 • Potassium chloride solution (KCl; 1 M; Sigma-Aldrich, synthesized and HPLC-purified from IDT): QP...

©

cat. no. 60142-100ML-F) CACCGA-3′ and QP2: 5′-CAAGCAGAAGACGGCATACGA-3′

• Glycerol (Sigma-Aldrich, cat. no. G5516-100ML) • High-sensitivity next-generation sequencing (NGS) fragment analysis kit

• Protease (7.5 Anson units (AU); Qiagen, cat. no. 19155) (Advanced Analytical Technologies, cat. no. DNF-486-0500)

• Nuclease-free water (Ambion, cat. no. AM9932) • 40% Acryl/Bis (29:1; Amresco, cat. no. 0311-1L) ! CAUTION This compound

• MspI (10 U/µl; Thermo Scientific, cat. no. ER0541) is harmful if swallowed or inhaled, and it causes irritation to the skin, the

• Tango buffer (10×; Thermo Scientific, cat. no. BY5) eyes and the respiratory tract. Handle it using appropriate equipment.

• λ-DNA (dam–, dcm–; 0.3 µg/µl, Thermo Scientific, cat. no. SD0021) • TBE buffer (5×; Amresco, cat. no. J885-1L)

• Klenow fragment (exo–, 5 U/µl; Thermo Scientific, cat. no. EP0422) • Ammonium persulfate (AP; Sigma-Aldrich, cat. no. A3678-100G)

• dATP (100 mM; New England BioLabs, cat. no. N0440S) ! CAUTION AP is harmful if swallowed or inhaled, and it causes irritation

• dCTP (100 mM; New England BioLabs, cat. no. N0441S) to the skin, the eyes and the respiratory tract. Handle it using appropriate

• dGTP (100 mM; New England BioLabs, cat. no. N0442S) equipment.

• T4 DNA ligase (highly concentrated; 30 Weiss U/µl; Thermo Scientific, • N,N,N′,N′-tetramethylethylenediamine (TEMED; Sigma-Aldrich,

cat. no. EL0013) cat. no. T9281-100ML) ! CAUTION TEMED is harmful if swallowed or

• ATP (100 mM; Thermo Scientific, cat. no. R0441) inhaled, and it causes irritation to the skin, the eyes and the respiratory

• Adapters, part of the Illumina TruSeq DNA sample preparation kits tract. Handle it using appropriate equipment.

(Illumina, cat. no. FC-121-2001)  CRITICAL Illumina TruSeq DNA • Ammonium acetate (Amresco, cat. no. 0103-500G)

adapters are highly recommended. If they are not available, ensure that the • Magnesium acetate tetrahydrate (Amresco, cat. no. 0131-500G)

cytosines (Cs) of the ordered oligos are methyl group–modified (mCs) and • SDS (Amresco, cat. no. S0227-500G)

double HPLC-purified. The oligo sequences of these indexed adapters are • EDTA (Amresco, cat. no. 0322-1KG)

listed in Table 1. • PBS buffer (pH 7.2; 1×; Gibco, cat. no. 14249-95)

• MethyCode bisulfite conversion kit (50 reactions; Thermo Scientific, • Leukemia inhibitory factor (LIF; 10 million units/1 ml, Millipore,

cat. no. MECOV-50) cat. no. ESG1107)

• tRNA (Roche, cat. no. 10109517001) • DMEM: nutrient mixture F-12 (DMEM/F-12; Gibco, cat. no. 11320-033)

• PfuTurbo Cx hotstart DNA polymerase (2.5 U/µl; Agilent Technologies, • FBS (Gibco, cat. no. 12483-020)

cat. no. 600412) • Trypsin-EDTA, 0.05% (wt/vol) (1×; Gibco, cat. no. 25300-062)

• dNTP mix (10 mM each; Clontech, cat. no. D4030RA) • Albumin, acetylated from bovine serum (Ac-BSA; Sigma-Aldrich,

• Agencourt AMPure XP beads (Beckman Coulter; cat. no. A63881) cat. no. B8894)

nature protocols | VOL.10 NO.5 | 2015 | 647

## Page 4

protocol

Table 1 | Oligo sequences of adapters.