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

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## Page 14

protocol

Figure 4 \| Box plots of the mapping efficiencies a b
and the total unique CpG sites covered in our 80 25
scRRBS data. (a) Box plot of the mapping
efficiencies of some scRRBS libraries. The six 20
items on the left in a indicate different types of 60
human cells, including single human sperm cells 15
(n = 4), single metaphase II oocytes (n = 2), 40
single female pronuclei (n = 11), single male 10
pronuclei (n = 11), 20–200 pooled blastomeres of
human preimplantation embryos (n = 6) and bulk 20
5
hESCs (human embryonic stem cells; n = 2). The
seven items (excluding the negative controls) on 0 0
the right in a represent different types of
mouse cells, including single mouse sperm cells
(n = 4), single metaphase II oocytes (n = 2),
single female pronuclei (n = 4), single male
pronuclei (n = 4), single mESCs (n = 8), 5–20
pooled mESCs (n = 3) and bulk mESCs (n = 2) as
control. (b) Box plot of the total unique CpG sites
covered in our scRRBS data. The four items on the
left in b represent four different types of human Human cells Mouse cells Human cells Mouse cells
cells, including single sperm cells (haploid,
n = 4), single metaphase II oocytes (with polar bodies removed, diploid, n = 2), single female pronuclei (haploid, n = 11) and single male pronuclei (haploid,
n = 11). The six items on the right in b indicate different types of mouse cells, including single mESCs (diploid, n = 8), single sperm cells (haploid, n = 4),
single metaphase II oocytes (with polar bodies removed, diploid, n = 2), single female pronuclei (haploid, n = 4), single male pronuclei (haploid, n = 4)
and bulk mESCs (n = 2), respectively. Middle lines in the box indicate the median values, edges and whiskers of the box indicate the 25th/75th percentiles,
and the 2.5th/97.5th percentiles, respectively. Some extreme values outside of the whisker boundaries are considered outliers.


Figure 5 \| The methylation status of a representative locus of sperm-specific chr1: 1,098,913–1,100,078 (1,166 bp)
differentially methylated regions (DMRs). The methylation levels of most MII oocyte-#1
of the CpG sites at this locus in the four single human sperm cells are fully MII oocyte-#2
methylated (black filled circles), and most of the CpG sites at this locus MII oocyte-#3
in the three human metaphase II oocytes are unmethylated (white open
circles). The circles in the bulk hESC track indicate the CpG sites covered in Sperm-#1
the bulk hESC RRBS sample, with DNA methylation levels ranging from 0% to Sperm-#2
100% (color key: white to black, respectively). The filled brown circles in the
bottom track represent all of the CpG sites at this genomic locus. Sperm-#3

Sperm-#4
The majority of the covered CpG sites should show digitized Bulk_hESC
DNA methylation; i.e., they are either fully methylated or Genomic track
unmethylated (Fig. 5; supplementary Figs. 2 and 3).
Plotting the scRRBS data of individual cells across genes shows that methylation levels are high on gene bodies compared
with neighboring genomic regions, and that there is an expected hypomethylation valley around the transcriptional
start sites (TSSs) (Fig. 6). Moreover, methylation levels gradually increase from the 5′ end (TSS side) to the 3′ end
(transcriptional end site (TES) side) of the gene body. The scRRBS technique is able to reveal global demethylation of the
maternal and paternal genomes during zygotic development (Fig. 6; supplementary Figs. 4 and 5), in which the paternal
genome is demethylated much faster than the maternal genome in human zygotes, a finding consistent with previous
immunofluorescence analysis10.
a MII oocyte (n = 3) b Sperm (n = 4)
PN 9–11 h after ICSI (n = 3) PN 9–11 h after ICSI (n = 2)
PN 14–15 h after ICSI (n = 3) PN 14–15 h after ICSI (n = 3)
PN 18–22 h after ICSI (n = 3) PN 18–22 h after ICSI (n = 3) Figure 6 \| The average DNA methylation levels
80 PN 25–28 h after ICSI (n = 5) 80 PN 25–28 h after ICSI (n = 5)
across gene bodies and the flanking intergenic
regions. (a,b) Average DNA methylation levels
along the transcripts and 15 kb upstream
60 60 and downstream of the TSSs and the TESs of
all RefSeq genes in the scRRBS data set of
40 40 human single metaphase II oocytes and single
female pronuclei at different time points after
intracytoplasmic sperm injection (ICSI) (a),
20 20 as well as in the scRRBS data set of human
single sperm cells and single male pronuclei at
different time points after ICSI (b). This shows
Down Gene body Up Down Gene body Up global demethylation patterns in the male and
0 0
–15 kb0% 20% 40% 60% 80% 100%15 kb –15 kb0% 20% 40% 60% 80% 100%15 kb female pronuclei on gene bodies and neighboring

TSS TES TSS TES intergenic regions.

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

## Page 15

protocol

Note: Any Supplementary Information and Source Data files are available in the 21. Gifford, C.A. et al. Transcriptional and epigenetic dynamics during
online version of the paper. specification of human embryonic stem cells. Cell 153, 1149–1163 (2013).
22. Ziller, M.J. et al. Charting a dynamic DNA methylation landscape of the
acknowle DGMents We thank J. Qiao and L. Yan for their great help. human genome. Nature 500, 477–481 (2013).
The project was supported by the National Science Foundation of China 23. Lister, R. et al. Global epigenomic reconfiguration during mammalian brain
(31322037 and 31271543) and the National Basic Research Program of China development. Science 341, 1237905 (2013).
(2012CB966704 and 2011CB966303). This work is supported by a collaborative 24. Wen, L. et al. Whole-genome analysis of 5-hydroxymethylcytosine and
grant from the Center for Molecular and Translational Medicine. 5-methylcytosine at base resolution in the human brain. Genome Biol. 15,
R49 (2014).
aut Hor contr IButIons L.W. and F.T. conceived the experiments and 25. Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and
supervised the project. H.G., F.G., X.L., X.W. and X.F. carried out all of the differentiated cells. Nature 454, 766–770 (2008).
experiments. P.Z. conducted the bioinformatic analyses. H.G., P.Z., L.W. and 26. Meissner, A. et al. Reduced representation bisulfite sequencing for
F.T. wrote the manuscript with contributions from all of the authors. comparative high-resolution DNA methylation analysis. Nucleic Acids Res.
33, 5868–5877 (2005).
coMpetInG FInanc Ial Interests The authors declare no competing financial 27. Gu, H. et al. Preparation of reduced representation bisulfite sequencing
interests. libraries for genome-scale DNA methylation profiling. Nat. Protoc. 6,

## Page 14

protocol

Figure 4 | Box plots of the mapping efficiencies a b

and the total unique CpG sites covered in our 80 25

scRRBS data. (a) Box plot of the mapping

efficiencies of some scRRBS libraries. The six 20

items on the left in a indicate different types of 60

human cells, including single human sperm cells 15

(n = 4), single metaphase II oocytes (n = 2), 40

single female pronuclei (n = 11), single male 10

pronuclei (n = 11), 20–200 pooled blastomeres of

human preimplantation embryos (n = 6) and bulk 20

5

hESCs (human embryonic stem cells; n = 2). The

seven items (excluding the negative controls) on 0 0

the right in a represent different types of

mouse cells, including single mouse sperm cells

(n = 4), single metaphase II oocytes (n = 2),

single female pronuclei (n = 4), single male

pronuclei (n = 4), single mESCs (n = 8), 5–20

pooled mESCs (n = 3) and bulk mESCs (n = 2) as

control. (b) Box plot of the total unique CpG sites

covered in our scRRBS data. The four items on the

left in b represent four different types of human Human cells Mouse cells Human cells Mouse cells

cells, including single sperm cells (haploid,

n = 4), single metaphase II oocytes (with polar bodies removed, diploid, n = 2), single female pronuclei (haploid, n = 11) and single male pronuclei (haploid,

n = 11). The six items on the right in b indicate different types of mouse cells, including single mESCs (diploid, n = 8), single sperm cells (haploid, n = 4),

single metaphase II oocytes (with polar bodies removed, diploid, n = 2), single female pronuclei (haploid, n = 4), single male pronuclei (haploid, n = 4)

and bulk mESCs (n = 2), respectively. Middle lines in the box indicate the median values, edges and whiskers of the box indicate the 25th/75th percentiles,

and the 2.5th/97.5th percentiles, respectively. Some extreme values outside of the whisker boundaries are considered outliers.

Figure 5 | The methylation status of a representative locus of sperm-specific chr1: 1,098,913–1,100,078 (1,166 bp)

differentially methylated regions (DMRs). The methylation levels of most MII oocyte-#1

of the CpG sites at this locus in the four single human sperm cells are fully MII oocyte-#2

methylated (black filled circles), and most of the CpG sites at this locus MII oocyte-#3

in the three human metaphase II oocytes are unmethylated (white open

circles). The circles in the bulk hESC track indicate the CpG sites covered in Sperm-#1

the bulk hESC RRBS sample, with DNA methylation levels ranging from 0% to Sperm-#2

100% (color key: white to black, respectively). The filled brown circles in the

bottom track represent all of the CpG sites at this genomic locus. Sperm-#3

Sperm-#4

The majority of the covered CpG sites should show digitized Bulk_hESC

DNA methylation; i.e., they are either fully methylated or Genomic track

unmethylated (Fig. 5; supplementary Figs. 2 and 3).

Plotting the scRRBS data of individual cells across genes shows that methylation levels are high on gene bodies compared

with neighboring genomic regions, and that there is an expected hypomethylation valley around the transcriptional

start sites (TSSs) (Fig. 6). Moreover, methylation levels gradually increase from the 5′ end (TSS side) to the 3′ end

(transcriptional end site (TES) side) of the gene body. The scRRBS technique is able to reveal global demethylation of the

maternal and paternal genomes during zygotic development (Fig. 6; supplementary Figs. 4 and 5), in which the paternal

genome is demethylated much faster than the maternal genome in human zygotes, a finding consistent with previous

immunofluorescence analysis10.

a MII oocyte (n = 3) b Sperm (n = 4)

PN 9–11 h after ICSI (n = 3) PN 9–11 h after ICSI (n = 2)

PN 14–15 h after ICSI (n = 3) PN 14–15 h after ICSI (n = 3)

PN 18–22 h after ICSI (n = 3) PN 18–22 h after ICSI (n = 3) Figure 6 | The average DNA methylation levels

80 PN 25–28 h after ICSI (n = 5) 80 PN 25–28 h after ICSI (n = 5)

across gene bodies and the flanking intergenic

regions. (a,b) Average DNA methylation levels

along the transcripts and 15 kb upstream

60 60 and downstream of the TSSs and the TESs of

all RefSeq genes in the scRRBS data set of

40 40 human single metaphase II oocytes and single

female pronuclei at different time points after

intracytoplasmic sperm injection (ICSI) (a),

20 20 as well as in the scRRBS data set of human

single sperm cells and single male pronuclei at

different time points after ICSI (b). This shows

Down Gene body Up Down Gene body Up global demethylation patterns in the male and

0 0

–15 kb0% 20% 40% 60% 80% 100%15 kb –15 kb0% 20% 40% 60% 80% 100%15 kb female pronuclei on gene bodies and neighboring

TSS TES TSS TES intergenic regions.

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

## Page 15

protocol

Note: Any Supplementary Information and Source Data files are available in the 21. Gifford, C.A. et al. Transcriptional and epigenetic dynamics during

online version of the paper. specification of human embryonic stem cells. Cell 153, 1149–1163 (2013).

22. Ziller, M.J. et al. Charting a dynamic DNA methylation landscape of the

acknowle DGMents We thank J. Qiao and L. Yan for their great help. human genome. Nature 500, 477–481 (2013).

The project was supported by the National Science Foundation of China 23. Lister, R. et al. Global epigenomic reconfiguration during mammalian brain

(31322037 and 31271543) and the National Basic Research Program of China development. Science 341, 1237905 (2013).

(2012CB966704 and 2011CB966303). This work is supported by a collaborative 24. Wen, L. et al. Whole-genome analysis of 5-hydroxymethylcytosine and

grant from the Center for Molecular and Translational Medicine. 5-methylcytosine at base resolution in the human brain. Genome Biol. 15,

R49 (2014).

aut Hor contr IButIons L.W. and F.T. conceived the experiments and 25. Meissner, A. et al. Genome-scale DNA methylation maps of pluripotent and

supervised the project. H.G., F.G., X.L., X.W. and X.F. carried out all of the differentiated cells. Nature 454, 766–770 (2008).

experiments. P.Z. conducted the bioinformatic analyses. H.G., P.Z., L.W. and 26. Meissner, A. et al. Reduced representation bisulfite sequencing for

F.T. wrote the manuscript with contributions from all of the authors. comparative high-resolution DNA methylation analysis. Nucleic Acids Res.

33, 5868–5877 (2005).

coMpetInG FInanc Ial Interests The authors declare no competing financial 27. Gu, H. et al. Preparation of reduced representation bisulfite sequencing

interests. libraries for genome-scale DNA methylation profiling. Nat. Protoc. 6,