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

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 CRITICAL STEP There will be some residual primer dimers even after the gel purification, necessitating this AMPure XP
beads purification step.
? TROUBLESHOOTING
 PAUSE POINT The final eluate could be stored at −20 °C for no longer than 3 months.reserved.
rights
All
Inc. a b
Marker #1 #2 #3 Marker 9,760
9,725 LM Single mESC RRBS UM 6,000
9,700 3,000
700 bp 2,000America, 9,675 ...
1,200
9,650 1,000900
800
9,625 700
200 bp
9,600 600Nature
100 bp RFU 9,575 225 500
9,550 211 4002015 9,525
© 9,500 300 ...
9,475
200
9,450

9,425
100
9,400
9,378 35
35 100 200 300 400 500 600 7008009001,0001,2001,5002,000 3,000 6,000
Size (bp)
c
9,820
9,800
Figure 2 \| The size distribution of LM Single mouse pronucleus RRBS UM 6,000
9,750 3,000
the typical scRRBS libraries. (a) The 2,000

1,200 polyacrylamide TBE gel results of 9,700 ...
three scRRBS libraries (before 800900
9,650 227 700 gel-based size selection); the DNA is ...
smeared from 200 bp to ~5 kb, with 9,600 500 RFU some detectable adapter dimers at ...
~120 bp. (b,c) The Fragment Analyzer
results of two scRRBS libraries 9,500 300
372 established from a single mESC (b)
9,450 200
and a single mouse pronucleus (c).
Typical DNA size distribution in 9,400 100
scRRBS libraries ranges from 160
to 350 bp, with visible peaks 9,344 35
35 100 200 300 400 500 600 700 800900 corresponding to MspI fragments ...
for some repetitive elements. Size (bp)


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

## Page 11

protocol

Box 2 \| ‘Crush and soak’ method ● TIMING 12–14 h

1. Use a needle from a 1-ml syringe to make several holes in the bottom of 0.5-ml thin-walled PCR tubes.
 CRITICAL STEP Handle the needle with appropriate protection.
2. Transfer the gel slices into 0.5-ml thin-walled PCR tubes, place the tubes into 1.5-ml Eppendorf DNA LoBind tubes and centrifuge
them at 13,000g for at least 1 min at room temperature until the gel slice collects in the bottom of the 1.5-ml tubes.
3. Add diffusion buffer (~350 µl, Reagent Setup) to the 1.5-ml tubes until all the gel debris is covered with buffer.
4. Incubate the slurry on a Thermomixer by shaking for 2–12 h at 50 °C. Longer incubations give better recoveries.
 CRITICAL STEP The slurry should be incubated for no less than 2 h.
5. Transfer the eluate and the gel debris to the top of a 10-µm filter spin, and then centrifuge the filter at 3,000g for 0.5–1 min at
room temperature to ensure that all of the eluate passes through the filter and flows to the bottom of the collection tubes.
6. Transfer the flow-through to new 1.5-ml Eppendorf DNA LoBind tubes.
 CRITICAL STEP The flow-through should not be discarded.



Quality control and high-throughput DNA sequencing ● TIMING 10–18 d
42\| Quantify the final single-cell RRBS library from Step 41 with a Qubit fluorometer and the Qubit dsDNA HS assay kit.
Use the qPCR assay to determine the concentration of each single-cell RRBS sample.
 CRITICAL STEP The typical yield of the scRRBS libraries is ~20–30 ng (using the Qubit fluorometer for quantification) after
gel-based size selection and AMPure XP beads purification, with <1 ng in the pick-buffer-only negative controls.reserved.  CRITICAL STEP The standard curve–based qPCR assay is a standard quantification assessment for the Illumina libraries
rights beforecurve; ondeepthesequencing.basis of thisFirst,curvepreparethe numberserial oftenfoldadapter-insert-adapterdilutions of the standardmoleculesIlluminaof thelibrariesscRRBS librariesto generatecanthebe determined.standard
All
43\| Assess the final libraries using a Fragment Analyzer (Advanced Analytical Technologies) to check the size distributions
Inc. (Fig. 2b).
 CRITICAL STEP If available, an Agilent Bioanalyzer 2100 can be used as an alternative to the Fragment Analyzer to
evaluate the quality and the size distributions of the final libraries.
 CRITICAL STEP Typical DNA size distribution in scRRBS libraries ranges from 160 to 350 bp, with visible peaksAmerica,
corresponding to MspI fragments for some repetitive elements (Fig. 2b,c). If the Fragment Analyzer results show that
there are still primer dimers present, perform another clean-up step with 1:1-fold AMPure XP beads as described in Box 1.Nature
2015 44\| Sequence the libraries using HiSeq 2000/2500 sequencers with cluster densities at 75–85% of that used in regular bulk
© DNA or RNA sequencing. Raw data

Data analysis for single-cell RRBS data ● TIMING 2–3 d Step 1: quality control (Step 45)
 CRITICAL An overview of the major procedures involved
in scRRBS data analysis is summarized in Figure 3. An Clean data
archive containing custom scripts used in this protocol is
available as Supplementary Data. The overview of these Step 2: alignment (Step 46)
custom scripts, including the script names, functions and
the corresponding protocol steps in which they should be SAM file
used, is listed in Table 2.

 CRITICAL STEP There will be some residual primer dimers even after the gel purification, necessitating this AMPure XP

beads purification step.

? TROUBLESHOOTING

 PAUSE POINT The final eluate could be stored at −20 °C for no longer than 3 months.reserved.

rights

All

Inc. a b

Marker #1 #2 #3 Marker 9,760

9,725 LM Single mESC RRBS UM 6,000

9,700 3,000

700 bp 2,000America, 9,675 ...

1,200

9,650 1,000900

800

9,625 700

200 bp

9,600 600Nature

100 bp RFU 9,575 225 500

9,550 211 4002015 9,525

9,475

200

9,450

9,425

100

9,400

9,378 35

35 100 200 300 400 500 600 7008009001,0001,2001,5002,000 3,000 6,000

Size (bp)

c

9,820

9,800

Figure 2 | The size distribution of LM Single mouse pronucleus RRBS UM 6,000

9,750 3,000

the typical scRRBS libraries. (a) The 2,000

1,200 polyacrylamide TBE gel results of 9,700 ...

three scRRBS libraries (before 800900

9,650 227 700 gel-based size selection); the DNA is ...

smeared from 200 bp to ~5 kb, with 9,600 500 RFU some detectable adapter dimers at ...

~120 bp. (b,c) The Fragment Analyzer

results of two scRRBS libraries 9,500 300

372 established from a single mESC (b)

9,450 200

and a single mouse pronucleus (c).

Typical DNA size distribution in 9,400 100

scRRBS libraries ranges from 160

to 350 bp, with visible peaks 9,344 35

35 100 200 300 400 500 600 700 800900 corresponding to MspI fragments ...

for some repetitive elements. Size (bp)

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

## Page 11

protocol

Box 2 | ‘Crush and soak’ method ● TIMING 12–14 h

1. Use a needle from a 1-ml syringe to make several holes in the bottom of 0.5-ml thin-walled PCR tubes.

 CRITICAL STEP Handle the needle with appropriate protection.

2. Transfer the gel slices into 0.5-ml thin-walled PCR tubes, place the tubes into 1.5-ml Eppendorf DNA LoBind tubes and centrifuge

them at 13,000g for at least 1 min at room temperature until the gel slice collects in the bottom of the 1.5-ml tubes.

3. Add diffusion buffer (~350 µl, Reagent Setup) to the 1.5-ml tubes until all the gel debris is covered with buffer.

4. Incubate the slurry on a Thermomixer by shaking for 2–12 h at 50 °C. Longer incubations give better recoveries.

 CRITICAL STEP The slurry should be incubated for no less than 2 h.

5. Transfer the eluate and the gel debris to the top of a 10-µm filter spin, and then centrifuge the filter at 3,000g for 0.5–1 min at

room temperature to ensure that all of the eluate passes through the filter and flows to the bottom of the collection tubes.

6. Transfer the flow-through to new 1.5-ml Eppendorf DNA LoBind tubes.

 CRITICAL STEP The flow-through should not be discarded.

Quality control and high-throughput DNA sequencing ● TIMING 10–18 d

42| Quantify the final single-cell RRBS library from Step 41 with a Qubit fluorometer and the Qubit dsDNA HS assay kit.

Use the qPCR assay to determine the concentration of each single-cell RRBS sample.

 CRITICAL STEP The typical yield of the scRRBS libraries is ~20–30 ng (using the Qubit fluorometer for quantification) after

gel-based size selection and AMPure XP beads purification, with <1 ng in the pick-buffer-only negative controls.reserved.  CRITICAL STEP The standard curve–based qPCR assay is a standard quantification assessment for the Illumina libraries

rights beforecurve; ondeepthesequencing.basis of thisFirst,curvepreparethe numberserial oftenfoldadapter-insert-adapterdilutions of the standardmoleculesIlluminaof thelibrariesscRRBS librariesto generatecanthebe determined.standard

All

43| Assess the final libraries using a Fragment Analyzer (Advanced Analytical Technologies) to check the size distributions

Inc. (Fig. 2b).

 CRITICAL STEP If available, an Agilent Bioanalyzer 2100 can be used as an alternative to the Fragment Analyzer to

evaluate the quality and the size distributions of the final libraries.

 CRITICAL STEP Typical DNA size distribution in scRRBS libraries ranges from 160 to 350 bp, with visible peaksAmerica,

corresponding to MspI fragments for some repetitive elements (Fig. 2b,c). If the Fragment Analyzer results show that

there are still primer dimers present, perform another clean-up step with 1:1-fold AMPure XP beads as described in Box 1.Nature

2015 44| Sequence the libraries using HiSeq 2000/2500 sequencers with cluster densities at 75–85% of that used in regular bulk

Data analysis for single-cell RRBS data ● TIMING 2–3 d Step 1: quality control (Step 45)

 CRITICAL An overview of the major procedures involved

in scRRBS data analysis is summarized in Figure 3. An Clean data

archive containing custom scripts used in this protocol is

available as Supplementary Data. The overview of these Step 2: alignment (Step 46)

custom scripts, including the script names, functions and

the corresponding protocol steps in which they should be SAM file

used, is listed in Table 2.