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

text stringlengths 0 4.18k
of specific studies. sis prov ides information about the average methylation level for
Applications of scRRBS each covered CpG site w ithin the population, which is similar to
The advantage of the scRRBS method is its applicabilit y to sub- conventional RRBS. Alternatively, if you wish to study cell hetero-
nanog ram levels of DNA as star ting mater ial, dow n to a sing le geneity w ith respect to DNA methylation w ithin a population, or
cell. This is par ticularly useful when the star ting mater ials are if there are only one or a few highly heterogeneous cells within the
ver y limited and precious, such as mammalian early embr yos and biological sample, indiv idual cells must be analyzed; in this case,
primordial germ cells. It also enables the heterogeneit y of DNA dozens (10–100) of sing le cells are required to obtain accurate
methylomes among individual cells to be studied, which may have measurement of DNA methylation for a cer tain cell t y pe. This
important roles in biological processes such as cell differentiation, analysis detects the methylation status of covered CpG sites in
memor y formation and oncogenesis. As examples, we have suc- a digitized manner, as each indiv idual mammalian cell contains
cessfully applied scRRBS to individual mouse sperm cells, and we only two copies of DNA molecules, or one copy of DNA molecule
detected each recovered CpG site as either fully unmethylated or when a haploid germ cell is analyzed.

fully methylated, which is expected because sperm cells contain Cont rols. It is impor tant to avoid any DNA contamination in
only a sing le copy of the haploid genome17. We have also applied scRRBS experiments. All reagents and consumables used in this
this approach to indiv idual human and mouse pronuclei iso- protocol should be free of exogenous DNA, and ‘picking-buffer-
lated from zygotes, and we obser ved g lobal 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 complexit y of
contamination during the whole experimental process. scRRBS libraries.
Enzy me choice. The restriction endonuclease MspI (C↓CGG) is D a t a a n a l y s i s . Bioinfor mat ics analysis me tho ds 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 repor ted prev iously, which are designed to
however, other restriction endonucleases such as TaqI ( T↓CGA) map bisulfite-conver ted sequence reads to a reference genome
could be chosen to capture different genomic reg ions. In addi- and to deter mine cy tosine methylation states at sing le-base
tion, digestion using two or more endonucleases may increase resolution10,17. Customized scr ipts w r itten in Perl or Py thon
the genomic coverage. can be applied for discarding low-qualit y reads, t r imming
adapter sequences and remov ing additional bases that are ar ti-
Sequencing. Different sequencing platforms can be used, includ- ficially introduced dur ing the end-repair steps. Alter natively,
ing the Illumina HiSeq 2000 and HiSeq 2500 platforms we use. the Tr im Galore tool can be used (http://www.bioinformatics.
We gener al ly sequence each scRRBS libr ar y to ~10 mil lion 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 w ill only slig htly raise the genomic coverage. alignment of the bisulfite-conver ted sequences and dow nstream
In our exper ience, a cluster densit y of 80% of the regular or analysis such as v isualization of the data, compar ison between
mixing scRRBS libr ar ies w ith other non-bisulfite-sequencing samples and identification of differentially methylated reg ions.
librar ies w ill increase the y ield and qualit y of the reads, which The Bismark program works well in our hands.






Mater Ials
REAGENTS •Phusion high-fidelity (HF) PCR master mix with HF buffer (New England
 cr ItIcal All reagents used in this experiment must be free of nucleases. BioLabs, cat. no. M0531S)
! caut Ion 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) ! caut Ion 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 respiratory
•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) ! caut Ion 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)
the eyes and the respiratory tract. Handle it using appropriate equipment. •Ethanol (Sigma-Aldrich, cat. no. E7023) ! caut Ion Ethanol is flammable.
 cr ItIcal 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 be
•Potassium chloride solution (KCl; 1 M; Sigma-Aldrich, synthesized and HPLC-purified from IDT): QP1: 5′-AATGATACGGCGAC
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) ! caut Ion 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) ! caut Ion 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) ! caut Ion 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)  cr ItIcal 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.

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

Applications of scRRBS each covered CpG site w ithin the population, which is similar to

The advantage of the scRRBS method is its applicabilit y to sub- conventional RRBS. Alternatively, if you wish to study cell hetero-

nanog ram levels of DNA as star ting mater ial, dow n to a sing le geneity w ith respect to DNA methylation w ithin a population, or

cell. This is par ticularly useful when the star ting mater ials are if there are only one or a few highly heterogeneous cells within the

ver y limited and precious, such as mammalian early embr yos and biological sample, indiv idual cells must be analyzed; in this case,

primordial germ cells. It also enables the heterogeneit y of DNA dozens (10–100) of sing le cells are required to obtain accurate

methylomes among individual cells to be studied, which may have measurement of DNA methylation for a cer tain cell t y pe. This

important roles in biological processes such as cell differentiation, analysis detects the methylation status of covered CpG sites in

memor y formation and oncogenesis. As examples, we have suc- a digitized manner, as each indiv idual mammalian cell contains

cessfully applied scRRBS to individual mouse sperm cells, and we only two copies of DNA molecules, or one copy of DNA molecule

detected each recovered CpG site as either fully unmethylated or when a haploid germ cell is analyzed.

fully methylated, which is expected because sperm cells contain Cont rols. It is impor tant to avoid any DNA contamination in

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

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

lated from zygotes, and we obser ved g lobal 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 complexit y of

contamination during the whole experimental process. scRRBS libraries.

Enzy me choice. The restriction endonuclease MspI (C↓CGG) is D a t a a n a l y s i s . Bioinfor mat ics analysis me tho ds 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 repor ted prev iously, which are designed to

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

could be chosen to capture different genomic reg ions. In addi- and to deter mine cy tosine methylation states at sing le-base

tion, digestion using two or more endonucleases may increase resolution10,17. Customized scr ipts w r itten in Perl or Py thon

the genomic coverage. can be applied for discarding low-qualit y reads, t r imming

adapter sequences and remov ing additional bases that are ar ti-

Sequencing. Different sequencing platforms can be used, includ- ficially introduced dur ing the end-repair steps. Alter natively,

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

We gener al ly sequence each scRRBS libr ar y to ~10 mil lion 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 w ill only slig htly raise the genomic coverage. alignment of the bisulfite-conver ted sequences and dow nstream

In our exper ience, a cluster densit y of 80% of the regular or analysis such as v isualization of the data, compar ison between

mixing scRRBS libr ar ies w ith other non-bisulfite-sequencing samples and identification of differentially methylated reg ions.

librar ies w ill increase the y ield and qualit y of the reads, which The Bismark program works well in our hands.

Mater Ials

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

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

! caut Ion 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) ! caut Ion 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 respiratory

•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) ! caut Ion 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)

the eyes and the respiratory tract. Handle it using appropriate equipment. •Ethanol (Sigma-Aldrich, cat. no. E7023) ! caut Ion Ethanol is flammable.

 cr ItIcal 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 be

•Potassium chloride solution (KCl; 1 M; Sigma-Aldrich, synthesized and HPLC-purified from IDT): QP1: 5′-AATGATACGGCGAC

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) ! caut Ion 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) ! caut Ion 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) ! caut Ion 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)  cr ItIcal 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.