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

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context in the λ-DNA genome are definitely unmethylated. The nonconversion rate of scRRBS is calculated as the number of
sequenced cytosines in non-CG contexts divided by all the covered cytosines in non-CG contexts in the λ-DNA genome.

47\| Once the alignment is complete, use SortSam.jar in the Picard toolkit to sort the mapping result in coordinate order to
prepare for the next step.
Example command:
java -Djava.io.tmpdir=<TMP> SortSam.jar I=<read1_val_1.fq.gz_bismark_pe.sam>
O=<read1_val_1.sort.bam> SORT_ORDER=coordinate VALIDATION_STRINGENCY=LENIENT
VERBOSITY=ERROR TMP_DIR=<TMP>
This command will generate a sorted BAM file.

48\| Create a pileup file of mapped data prepared for DNA methylation–level calculation. This step will give a pileup result for
each covered locus in the genome. Example command:

samtools mpileup -f <genome.fa> <read1_val_1.sort.bam> > <read1_val_1.pileup>

49\| Calculate the DNA methylation level for each covered CpG and non-CpG site. The DNA methylation level of each covered
cytosine is calculated as the number of reported C divided by the total number of reported C and T at the same genome
position. Example command:
Perl SingleC_MetLevel.pl <genome.fa> <read1_val_1.pileup> > <read1_val_1.SingleCmet>
The output file is tab-delimited, and it contains the chromosome, base position, reference genome, chain, total coverage
depth, number of methylated reads, number of unmethylated reads, methylation level and reference context (CpG, CHH or
CHG). This output file contains well-processed data that can be used to calculate the average DNA methylation of the
samples or the DNA methylation levels of any interesting annotated regions.







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

## Page 13

protocol


? trou Bles Hoot InG
Troubleshooting advice can be found in table 3.

taBle 3 \| Troubleshooting table.

step problem possible reason solution

7 Cell transfer failure Single cells may stick to the inside First, suck a small volume of PBA-BSA into the pipette, and then
wall of the mouth pipette pick the single cell into it. Ensure that the cell is already in the
pipette but near the tip of the pipette. Push all of the carryover
PBA-BSA out of the pipette, together with the cell

9 Incomplete cell lysis A single cell is not loaded into the During cell transfer, ensure that the cell is seeded into the
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 them
into small batches to avoid unnecessary freeze-thaw cycles,
especially for the primers

41 Excessive primer-dimer Too many PCR cycles or excessive Perform another round of AMPure XP bead purification to remove
contamination use of adapter or PCR primers contaminants. In addition, ensure that the concentrations of PCR
primers and PCR polymerase are appropriate


● tIMInG
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 h
Steps 16–19, adapter ligation: 9–10 h
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

ant IcIpate D 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

context in the λ-DNA genome are definitely unmethylated. The nonconversion rate of scRRBS is calculated as the number of

sequenced cytosines in non-CG contexts divided by all the covered cytosines in non-CG contexts in the λ-DNA genome.

47| Once the alignment is complete, use SortSam.jar in the Picard toolkit to sort the mapping result in coordinate order to

prepare for the next step.

Example command:

java -Djava.io.tmpdir=<TMP> SortSam.jar I=<read1_val_1.fq.gz_bismark_pe.sam>

O=<read1_val_1.sort.bam> SORT_ORDER=coordinate VALIDATION_STRINGENCY=LENIENT

VERBOSITY=ERROR TMP_DIR=<TMP>

This command will generate a sorted BAM file.

48| Create a pileup file of mapped data prepared for DNA methylation–level calculation. This step will give a pileup result for

each covered locus in the genome. Example command:

samtools mpileup -f <genome.fa> <read1_val_1.sort.bam> > <read1_val_1.pileup>

49| Calculate the DNA methylation level for each covered CpG and non-CpG site. The DNA methylation level of each covered

cytosine is calculated as the number of reported C divided by the total number of reported C and T at the same genome

position. Example command:

Perl SingleC_MetLevel.pl <genome.fa> <read1_val_1.pileup> > <read1_val_1.SingleCmet>

The output file is tab-delimited, and it contains the chromosome, base position, reference genome, chain, total coverage

depth, number of methylated reads, number of unmethylated reads, methylation level and reference context (CpG, CHH or

CHG). This output file contains well-processed data that can be used to calculate the average DNA methylation of the

samples or the DNA methylation levels of any interesting annotated regions.

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

## Page 13

protocol

? trou Bles Hoot InG

Troubleshooting advice can be found in table 3.

taBle 3 | Troubleshooting table.

step problem possible reason solution

7 Cell transfer failure Single cells may stick to the inside First, suck a small volume of PBA-BSA into the pipette, and then

wall of the mouth pipette pick the single cell into it. Ensure that the cell is already in the

pipette but near the tip of the pipette. Push all of the carryover

PBA-BSA out of the pipette, together with the cell

9 Incomplete cell lysis A single cell is not loaded into the During cell transfer, ensure that the cell is seeded into the

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 them

into small batches to avoid unnecessary freeze-thaw cycles,

especially for the primers

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

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

primers and PCR polymerase are appropriate

● tIMInG

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 h

Steps 16–19, adapter ligation: 9–10 h

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

ant IcIpate D 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