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

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lysis buffer, or the cell is not intact bottom of the tube. Centrifugation is crucial, and it cannot be
before transferring it by mouth omitted. In addition, ensure that the cells are of good
pipette morphology, and if possible always pick the healthiest cells.
The use of 5 µl of lysis buffer and 3 h of incubation is sufficient
to completely lyse the single cells

24, Low PCR amplification Too much DNA loss before PCR Avoid excessive pipetting, and use LoBind tubes during the whole
28 efficiency amplification procedure
Poor quality of PCR reagents Ensure that the reagents for PCR have not expired. Divide themreserved. into small batches to avoid unnecessary freeze-thaw cycles,
especially for the primers
rights 41 Excessive primer-dimer Too many PCR cycles or excessive Perform another round of AMPure XP bead purification to remove
All contamination use of adapter or PCR primers contaminants. In addition, ensure that the concentrations of PCR
primers and PCR polymerase are appropriate
Inc.

● TIMINGAmerica, Steps 1–9, cell culture, single-cell isolation and cell lysis: 4–5 d
Steps 10–12, MspI digestion: 3–4 h
Steps 13–15, end-repair/dA-tailing reaction: 1–2 hNature Steps 16–19, adapter ligation: 9–10 h
2015 Steps 20–23, bisulfite conversion: 3–4 h Steps 24–27, first-round PCR amplification: 3–4 h (plus purification time; see Box 1)
© Steps 28–31, second-round PCR amplification: 3–4 h (plus purification time; see Box 1)
Steps 32–41, size selection of the amplified DNA fragments: 9–10 h (plus purification time; see Box 1)
Steps 42–44, quality control and high-throughput DNA sequencing: 10–18 d
Steps 45–49, data analysis for single-cell RRBS data: 2–3 d
Box 1, DNA purification using AMPure XP beads: 0.5–1 h
Box 2, ‘Crush and soak’ method: 12–14 h

ANTICIPATED RESULTS
The yields of scRRBS libraries from different sample types (haploid cells or diploid cells) do not vary substantially. The
typical yield is ~20–30 ng (using a Qubit fluorometer for quantification) after gel-based size selection and AMPure XP beads
purification, with <1 ng in the ‘picking-buffer-only’ negative controls (using Qubit a fluorometer for quantification), and the
DNA fragment size in scRRBS libraries ranges from 160 to 350 bp, with visible peaks corresponding to the MspI fragments for
certain repetitive elements (Fig. 2b).
We have performed scRRBS on individual mouse and human metaphase II oocytes, sperm, male and female pronuclei of
zygotes, as well as individual mESCs10,17. The average mapping ratio of scRRBS is ~25%, which is lower than that observed
with standard RRBS, which ranges from 50 to 70%. This low mapping ratio may be due to the higher number of PCR amplifi
cation cycles required for scRRBS (Fig. 4).
For a mammalian individual diploid cell, scRRBS is expected to cover ~40% of the CpG sites (~1 million CpG sites in a
mouse and human diploid cell) that can be recovered by standard RRBS using thousands of cells10. Coverage lower than this
may be due to degradation of genomic DNA before cell lysis.
In our studies, only scRRBS samples with a high bisulfite conversion rate (>98%) were used for further analysis. We recov
ered between 0.2 and 1.5 million CpG sites from each individual haploid or diploid cell (Fig. 4 and Supplementary Fig. 1).


nature protocols \| VOL.10 NO.5 \| 2015 \| 657

## 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
105) 20 efficiencies of some scRRBS libraries. The six
items on the left in a indicate different types of (%) 60 (× human cells, including single human sperm cells ...
ratio (n = 4), single metaphase II oocytes (n = 2), 40 CpG
single female pronuclei (n = 11), single male 10 pronuclei (n = 11), 20–200 pooled blastomeres of Mapping ...
human preimplantation embryos (n = 6) and bulk 20 5 ...
seven items (excluding the negative controls) on 0 0
the right in a represent different types of
cells mouse cells, including single mouse sperm cells sperm oocyte ...
pronuclei (n = 4), single mESCs (n = 8), 5–20 Single female male Single female male Negative Single female ...
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)reserved. and bulk mESCs (n = 2), respectively. Middle lines in the box indicate the median values, edges and whiskers of the box indicate the 25th/75...
rights and the 2.5th/97.5th percentiles, respectively. Some extreme values outside of the whisker boundaries are considered outliers.
All 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
Inc. 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
in the three human metaphase II oocytes are unmethylated (white open MII oocyte-#3
circles). The circles in the bulk hESC track indicate the CpG sites covered in Sperm-#1America, 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-#3Nature Sperm-#4

Bulk_hESC majority of the covered CpG sites should show digitized...
© 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 MIIPNoocyte9–11 h(nafter= 3) ICSI (n = 3) b SpermPN 9–11(n =h4)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) ...
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
level level all RefSeq genes in the scRRBS data set of
human single metaphase II oocytes and single
40 40
female pronuclei at different time points after methylation ...
as well as in the scRRBS data set of human 20 20 DNA DNA ...
different time points after ICSI (b). This shows
0 Down Gene body Up 0 Down Gene body Up global demethylation patterns in the male and

lysis buffer, or the cell is not intact bottom of the tube. Centrifugation is crucial, and it cannot be

before transferring it by mouth omitted. In addition, ensure that the cells are of good

pipette morphology, and if possible always pick the healthiest cells.

The use of 5 µl of lysis buffer and 3 h of incubation is sufficient

to completely lyse the single cells

24, Low PCR amplification Too much DNA loss before PCR Avoid excessive pipetting, and use LoBind tubes during the whole

28 efficiency amplification procedure

Poor quality of PCR reagents Ensure that the reagents for PCR have not expired. Divide themreserved. into small batches to avoid unnecessary freeze-thaw cycles,

especially for the primers

rights 41 Excessive primer-dimer Too many PCR cycles or excessive Perform another round of AMPure XP bead purification to remove

All contamination use of adapter or PCR primers contaminants. In addition, ensure that the concentrations of PCR

primers and PCR polymerase are appropriate

Inc.

● TIMINGAmerica, Steps 1–9, cell culture, single-cell isolation and cell lysis: 4–5 d

Steps 10–12, MspI digestion: 3–4 h

Steps 13–15, end-repair/dA-tailing reaction: 1–2 hNature Steps 16–19, adapter ligation: 9–10 h

2015 Steps 20–23, bisulfite conversion: 3–4 h Steps 24–27, first-round PCR amplification: 3–4 h (plus purification time; see Box 1)

Steps 32–41, size selection of the amplified DNA fragments: 9–10 h (plus purification time; see Box 1)

Steps 42–44, quality control and high-throughput DNA sequencing: 10–18 d

Steps 45–49, data analysis for single-cell RRBS data: 2–3 d

Box 1, DNA purification using AMPure XP beads: 0.5–1 h

Box 2, ‘Crush and soak’ method: 12–14 h

ANTICIPATED RESULTS

The yields of scRRBS libraries from different sample types (haploid cells or diploid cells) do not vary substantially. The

typical yield is ~20–30 ng (using a Qubit fluorometer for quantification) after gel-based size selection and AMPure XP beads

purification, with <1 ng in the ‘picking-buffer-only’ negative controls (using Qubit a fluorometer for quantification), and the

DNA fragment size in scRRBS libraries ranges from 160 to 350 bp, with visible peaks corresponding to the MspI fragments for

certain repetitive elements (Fig. 2b).

We have performed scRRBS on individual mouse and human metaphase II oocytes, sperm, male and female pronuclei of

zygotes, as well as individual mESCs10,17. The average mapping ratio of scRRBS is ~25%, which is lower than that observed

with standard RRBS, which ranges from 50 to 70%. This low mapping ratio may be due to the higher number of PCR amplifi

cation cycles required for scRRBS (Fig. 4).

For a mammalian individual diploid cell, scRRBS is expected to cover ~40% of the CpG sites (~1 million CpG sites in a

mouse and human diploid cell) that can be recovered by standard RRBS using thousands of cells10. Coverage lower than this

may be due to degradation of genomic DNA before cell lysis.

In our studies, only scRRBS samples with a high bisulfite conversion rate (>98%) were used for further analysis. We recov

ered between 0.2 and 1.5 million CpG sites from each individual haploid or diploid cell (Fig. 4 and Supplementary Fig. 1).

nature protocols | VOL.10 NO.5 | 2015 | 657

## 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

105) 20 efficiencies of some scRRBS libraries. The six

items on the left in a indicate different types of (%) 60 (× human cells, including single human sperm cells ...

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

single female pronuclei (n = 11), single male 10 pronuclei (n = 11), 20–200 pooled blastomeres of Mapping ...

human preimplantation embryos (n = 6) and bulk 20 5 ...

seven items (excluding the negative controls) on 0 0

the right in a represent different types of

cells mouse cells, including single mouse sperm cells sperm oocyte ...

pronuclei (n = 4), single mESCs (n = 8), 5–20 Single female male Single female male Negative Single female ...

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

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

All 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

Inc. 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

in the three human metaphase II oocytes are unmethylated (white open MII oocyte-#3

circles). The circles in the bulk hESC track indicate the CpG sites covered in Sperm-#1America, 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-#3Nature Sperm-#4

Bulk_hESC majority of the covered CpG sites should show digitized...

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 MIIPNoocyte9–11 h(nafter= 3) ICSI (n = 3) b SpermPN 9–11(n =h4)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) ...

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

level level all RefSeq genes in the scRRBS data set of

human single metaphase II oocytes and single

40 40

female pronuclei at different time points after methylation ...

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

different time points after ICSI (b). This shows

0 Down Gene body Up 0 Down Gene body Up global demethylation patterns in the male and