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

text stringlengths 0 4.18k

Oligo name Sequence (all sequences read 5′–3′)

TruSeq universal adapter AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T

TruSeq adapter (index 1) GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 2) GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 3) GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 4) GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 5) GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 6) GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAATATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 7) GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 8) GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTGAATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 9) GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATCAGATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 10) GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGCTTATCTCGTATGCCGTCTTCTGCTTGreserved.
TruSeq adapter (index 11) GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCTACATCTCGTATGCCGTCTTCTGCTTG
rights TruSeq adapter (index 12) GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTAATCTCGTATGCCGTCTTCTGCTTG
All Index sequences are six bases, underlined.
The linkage of the last two bases of the TruSeq universal adapter oligo should be phosphorothiated to avoid nuclease cleavage of the T overhang.
Inc. The TruSeq adapter oligo (indexes 1–12) should be 5′-terminal phosphorylated for ligation of the inserted DNA fragments.


• DNA loading buffer (6×; Clontech, cat. no. 9156) • ChemiDoc XRS+ System with Image Lab Software (computer-connected,
• GeneRuler low-range DNA ladder (ready-to-use; Thermo Scientific, Bio-Rad, cat. no. 170-8265)America,
cat. no. SM1193) • 4-gel vertical electrophoresis system (Bio-Rad, cat. no.165-8006)
• DTT (Thermo Scientific, cat. no. R0861) • PowerPac basic power supply (Bio-Rad, cat. no. 164-5050)
• KAPA HiFi HotStart ReadyMix (2×; Kapa Biosystems, cat. no. KK2602) • Transference decoloring shaker (Qilinbeier, cat. no. TS-8)Nature EQUIPMENT • Fragment Analyzer (Advanced Analytical Technologies, ...
2015 •• PCRPCR tubes,tubes, 0.5-ml0.2-ml thin-walledthin-walled withwith flatflat capcap (Axygen,(Axygen, cat.cat. no.no. PCR-05-C)PCR-02-C) Software• Perl. Download freely from http://www.perl.org/
© • 1,000-µl universal fit filter tips (Axygen, cat. no. TF-1000-R-S) • Trim_galore (version 0.3.7). Freely available at http://www.bioinformatics.
• 200-µl universal fit filter tips (Axygen, cat. no. TF-200-R-S) babraham.ac.uk/projects/trim_galore/
• 100-µl universal fit filter tips (Axygen, cat. no. TF-100-R-S) • Bowtie (version 1, ref. 38). Download from http://bowtie-bio.sourceforge.
• 10-µl universal fit filter tips (Axygen, cat. no. TF-300-R-S) net/index.shtml
• DNA LoBind tubes, 1.5 ml (Eppendorf, cat. no. 022431021) • Bismark (version 0.7.6, ref. 35). Download from http://www.bioinformatics.
• DNA LoBind tubes, 2.0 ml (Eppendorf, cat. no. 022431048) babraham.ac.uk/projects/bismark/. Other similar tools can also be used
• Cell culture–treated dishes (60 mm; Thermo Scientific, cat. no. 130181) • Picard toolkit. Download from http://broadinstitute.github.io/picard/.
• Falcon conical centrifuge tubes (15 ml; Corning Life Sciences, Other similar tools can also be used
cat. no. 14-959-49D) • Samtools39. Download from http://samtools.sourceforge.net/
• Magnetic rack (Diagenode, cat. no. kch-816-001) • Java. Download from https://www.java.com/
• 10 µM filter spin (Zymo, cat. no. C1007-50) • Custom scripts. An archive of all customized scripts used in this protocol is
• Dark Reader transilluminator (Clare Chemical Research, cat. no. DR88X) available as Supplementary Data. Refer to Table 2 for details of each script
• Flaming/Brown micropipette puller (Sutter Instrument, P-1000) REAGENT SETUP
• Refrigerated microcentrifuge (5415R; Eppendorf, cat. no. 022621408) QP1, QP2 PCR primers Upon arrival, dissolve the forward (QP1) and
• Nonrefrigerated microcentrifuge (Heraeus Pico17; Thermo Scientific, reverse (QP2) primers using nuclease-free water to 100 µM as stock solutions
cat. no. 75002410) and dilute them to 10 µM as working solutions. Both the stock and working
• Thermomixer (Eppendorf, cat. no. 5355 000.011)
solutions are stable for 1 year if frozen at −80 °C.
• Qubit 2.0 fluorometer (Thermo Scientific, cat. no. Q32866)
Diffusion buffer Diffusion buffer is 500 mM ammonium acetate, 10 mM
• Borosilicate glass capillary (Sutter Instrument, cat. no. B100-58-10)
magnesium acetate, 1 mM EDTA (pH 8.0) and 1% (wt/vol) SDS. Diffusion ! CAUTION Be careful with the glass capillary; handle it with appropriate
buffer should be stored at room temperature for no longer than 6 months. protection.
PBS-BSA buffer Dilute Ac-BSA stock solution (20 mg/ml) with 1×PBS to • Aspirator tube assemblies for calibrated microcapillary pipettes
(Sigma-Aldrich, cat. no. A5177) 1 mg/ml as working solution and prepare aliquots in 1.5-ml Eppendorf DNA
• HL-2000 HybriLinker (UVP, cat. no. 95-0031-02) LoBind tubes. The working solution can be stored at −80 °C for at least
• Vortex (Scientific industries, cat. no. SI-0246) 6 months. BSA is essential to eliminate the nonspecific binding of single cells
• Thermocycler with a 96-well block (TProfessional Gradient 96; to the surface of the reaction tubes or glass capillaries.
Biometra, cat. no. 846-070-801) Protease, 20 mg/ml Soak 20 mg of Qiagen lyophilized protease in 1 ml of
• 16-tube magnetic rack (Diagenode, cat. no. B04000001 (kch-816-001)) 50% (vol/vol) glycerol and dissolve it for at least 30 min at 4 °C; next, prepare


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

## Page 5

protocol

Table 2 \| Customized scripts for data analysis.

Script name Functions Used in steps

01. trimQC.sh Quality control to filter out low-quality reads and to trim adapter sequences 45

02. Bismark_Genome_Preparation.sh Index the reference genome 46

03. Alignment.sh Read mapping using Bismark (version 0.7.6) 46

04. sort_pileup.sh Sort mapped SAM files and generate pileup files 48

05. SingleC_ MetLevel.sh DNA methylation level calculation of each covered cytosine 49




aliquots in 1.5-ml Eppendorf DNA LoBind tubes when fully dissolved. Store should be stored at a concentration >1 ng/µl at −20 °C for no longer than
the aliquots at −20 °C for no more than 6 months. 3 months and quantified and diluted just before use.
tRNA carrier Soak 10 mg of tRNA lyophilizate into 1 ml of nuclease-free CT conversion reagent Add 850 µl of nuclease-free water, 50 µl of
water to allow it to dissolve for at least 10 min, and then dilute it with resuspension buffer (part of the MethylCode bisulfite conversion kit) and
nuclease-free water to 10 ng/µl as the working concentration; divide the 300 µl of dilution buffer (part of the MethylCode bisulfite conversion kit)
solution into 1.5-ml Eppendorf DNA LoBind tubes and store the aliquots directly to the CT conversion powder (also part of the same kit). Mix the
at −80 °C for no more than 6 months. solution by brief intermittent vortexing until the solution becomes clear.
End-repair dNTP mix Mix and dilute the dATP, dGTP and dCTP stock Always keep the dissolved CT conversion reagent in the dark, and then storereserved. solutions (100 mM each) to 1 mM, 0.1 mM and 0.1 mM, respectively. Divide it for up to 2 weeks at −20 °C. Avoid repeated freeze/thaw cycles.

Oligo name Sequence (all sequences read 5′–3′)

TruSeq universal adapter AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T

TruSeq adapter (index 1) GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 2) GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 3) GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 4) GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 5) GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 6) GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAATATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 7) GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 8) GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTGAATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 9) GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATCAGATCTCGTATGCCGTCTTCTGCTTG

TruSeq adapter (index 10) GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGCTTATCTCGTATGCCGTCTTCTGCTTGreserved.

TruSeq adapter (index 11) GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCTACATCTCGTATGCCGTCTTCTGCTTG

rights TruSeq adapter (index 12) GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTAATCTCGTATGCCGTCTTCTGCTTG

All Index sequences are six bases, underlined.

The linkage of the last two bases of the TruSeq universal adapter oligo should be phosphorothiated to avoid nuclease cleavage of the T overhang.

Inc. The TruSeq adapter oligo (indexes 1–12) should be 5′-terminal phosphorylated for ligation of the inserted DNA fragments.

• DNA loading buffer (6×; Clontech, cat. no. 9156) • ChemiDoc XRS+ System with Image Lab Software (computer-connected,

• GeneRuler low-range DNA ladder (ready-to-use; Thermo Scientific, Bio-Rad, cat. no. 170-8265)America,

cat. no. SM1193) • 4-gel vertical electrophoresis system (Bio-Rad, cat. no.165-8006)

• DTT (Thermo Scientific, cat. no. R0861) • PowerPac basic power supply (Bio-Rad, cat. no. 164-5050)

• KAPA HiFi HotStart ReadyMix (2×; Kapa Biosystems, cat. no. KK2602) • Transference decoloring shaker (Qilinbeier, cat. no. TS-8)Nature EQUIPMENT • Fragment Analyzer (Advanced Analytical Technologies, ...

2015 •• PCRPCR tubes,tubes, 0.5-ml0.2-ml thin-walledthin-walled withwith flatflat capcap (Axygen,(Axygen, cat.cat. no.no. PCR-05-C)PCR-02-C) Software• Perl. Download freely from http://www.perl.org/

• 200-µl universal fit filter tips (Axygen, cat. no. TF-200-R-S) babraham.ac.uk/projects/trim_galore/

• 100-µl universal fit filter tips (Axygen, cat. no. TF-100-R-S) • Bowtie (version 1, ref. 38). Download from http://bowtie-bio.sourceforge.

• 10-µl universal fit filter tips (Axygen, cat. no. TF-300-R-S) net/index.shtml

• DNA LoBind tubes, 1.5 ml (Eppendorf, cat. no. 022431021) • Bismark (version 0.7.6, ref. 35). Download from http://www.bioinformatics.

• DNA LoBind tubes, 2.0 ml (Eppendorf, cat. no. 022431048) babraham.ac.uk/projects/bismark/. Other similar tools can also be used

• Cell culture–treated dishes (60 mm; Thermo Scientific, cat. no. 130181) • Picard toolkit. Download from http://broadinstitute.github.io/picard/.

• Falcon conical centrifuge tubes (15 ml; Corning Life Sciences, Other similar tools can also be used

cat. no. 14-959-49D) • Samtools39. Download from http://samtools.sourceforge.net/

• Magnetic rack (Diagenode, cat. no. kch-816-001) • Java. Download from https://www.java.com/

• 10 µM filter spin (Zymo, cat. no. C1007-50) • Custom scripts. An archive of all customized scripts used in this protocol is

• Dark Reader transilluminator (Clare Chemical Research, cat. no. DR88X) available as Supplementary Data. Refer to Table 2 for details of each script

• Flaming/Brown micropipette puller (Sutter Instrument, P-1000) REAGENT SETUP

• Refrigerated microcentrifuge (5415R; Eppendorf, cat. no. 022621408) QP1, QP2 PCR primers Upon arrival, dissolve the forward (QP1) and

• Nonrefrigerated microcentrifuge (Heraeus Pico17; Thermo Scientific, reverse (QP2) primers using nuclease-free water to 100 µM as stock solutions

cat. no. 75002410) and dilute them to 10 µM as working solutions. Both the stock and working

• Thermomixer (Eppendorf, cat. no. 5355 000.011)

solutions are stable for 1 year if frozen at −80 °C.

• Qubit 2.0 fluorometer (Thermo Scientific, cat. no. Q32866)

Diffusion buffer Diffusion buffer is 500 mM ammonium acetate, 10 mM

• Borosilicate glass capillary (Sutter Instrument, cat. no. B100-58-10)

magnesium acetate, 1 mM EDTA (pH 8.0) and 1% (wt/vol) SDS. Diffusion ! CAUTION Be careful with the glass capillary; handle it with appropriate

buffer should be stored at room temperature for no longer than 6 months. protection.

PBS-BSA buffer Dilute Ac-BSA stock solution (20 mg/ml) with 1×PBS to • Aspirator tube assemblies for calibrated microcapillary pipettes

(Sigma-Aldrich, cat. no. A5177) 1 mg/ml as working solution and prepare aliquots in 1.5-ml Eppendorf DNA

• HL-2000 HybriLinker (UVP, cat. no. 95-0031-02) LoBind tubes. The working solution can be stored at −80 °C for at least

• Vortex (Scientific industries, cat. no. SI-0246) 6 months. BSA is essential to eliminate the nonspecific binding of single cells

• Thermocycler with a 96-well block (TProfessional Gradient 96; to the surface of the reaction tubes or glass capillaries.

Biometra, cat. no. 846-070-801) Protease, 20 mg/ml Soak 20 mg of Qiagen lyophilized protease in 1 ml of

• 16-tube magnetic rack (Diagenode, cat. no. B04000001 (kch-816-001)) 50% (vol/vol) glycerol and dissolve it for at least 30 min at 4 °C; next, prepare

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

## Page 5

protocol

Table 2 | Customized scripts for data analysis.

Script name Functions Used in steps

01. trimQC.sh Quality control to filter out low-quality reads and to trim adapter sequences 45

02. Bismark_Genome_Preparation.sh Index the reference genome 46

03. Alignment.sh Read mapping using Bismark (version 0.7.6) 46

04. sort_pileup.sh Sort mapped SAM files and generate pileup files 48

05. SingleC_ MetLevel.sh DNA methylation level calculation of each covered cytosine 49

aliquots in 1.5-ml Eppendorf DNA LoBind tubes when fully dissolved. Store should be stored at a concentration >1 ng/µl at −20 °C for no longer than

the aliquots at −20 °C for no more than 6 months. 3 months and quantified and diluted just before use.

tRNA carrier Soak 10 mg of tRNA lyophilizate into 1 ml of nuclease-free CT conversion reagent Add 850 µl of nuclease-free water, 50 µl of

water to allow it to dissolve for at least 10 min, and then dilute it with resuspension buffer (part of the MethylCode bisulfite conversion kit) and

nuclease-free water to 10 ng/µl as the working concentration; divide the 300 µl of dilution buffer (part of the MethylCode bisulfite conversion kit)

solution into 1.5-ml Eppendorf DNA LoBind tubes and store the aliquots directly to the CT conversion powder (also part of the same kit). Mix the

at −80 °C for no more than 6 months. solution by brief intermittent vortexing until the solution becomes clear.

End-repair dNTP mix Mix and dilute the dATP, dGTP and dCTP stock Always keep the dissolved CT conversion reagent in the dark, and then storereserved. solutions (100 mM each) to 1 mM, 0.1 mM and 0.1 mM, respectively. Divide it for up to 2 weeks at −20 °C. Avoid repeated freeze/thaw cycles.