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• 40% Acryl/Bis (29:1; Amresco, cat. no. 0311-1L) ! CAUTI0N This compound is harmful if swallowed or inhaled, and it causes irritation to the skin, the eyes and the respiratory tract. Handle it using appropriate equipment. |
Enzyme choice. The restriction endonuclease MspI (C↓CGG) is commonly used in RRBS, and it is the focus of this scRRBS protocol. MspI digestion is strongly biased toward CGIs and promoters; however, other restriction endonucleases such as TaqI (T↓CGA) could be chosen to capture different genomic regions. In addition, di... |
Sequencing. Different sequencing platforms can be used, including the Illumina HiSeq 2000 and HiSeq 2500 platforms we use. We generally sequence each scRRBS library to ~10 million 100-bp paired-end reads. Increasing the sequencing depth to 20 million reads will only slightly raise the genomic coverage. In our experienc... |
## MATERIALS |
## REAGENTS |
▲ CRITICAL All reagents used in this experiment must be free of nucleases. ! CAUTION All samples from animals or humans should be processed according to the guidelines of the local government or institution. |
• Cell line or single cells of interest. In the PROCEDURE, we use as an example mESCs maintained in conventional DMEM, containing 20% (vol/vol) FBS in the presence of leukemia inhibitory factor (LIF) |
• Tris-EDTA solution (100×; Sigma-Aldrich, cat. no. T9285-100ML) |
• Triton X-100 (Sigma-Aldrich, cat. no. T8787-50ML) ! CAUTI0N Triton X-100 is harmful if swallowed or inhaled, and it causes irritation to the skin, the eyes and the respiratory tract. Handle it using appropriate equipment. ▲ CRITICAL Triton X-100 should be stored at room temperature (20–25 °C) for at most 1 year. |
• Potassium chloride solution (KCl; 1 M; Sigma-Aldrich, cat. no. 60142-100ML-F) |
• Glycerol (Sigma-Aldrich, cat. no. G5516-100ML) |
• Protease (7.5 Anson units (AU); Qiagen, cat. no. 19155) |
• Nuclease-free water (Ambion, cat. no. AM9932) |
• MspI (10 U/μl; Thermo Scientific, cat. no. ER0541) |
• Tango buffer (10×; Thermo Scientific, cat. no. BY5) |
• λ-DNA (dam-, dcm-; 0.3 µg/μl, Thermo Scientific, cat. no. SD0021) |
• Klenow fragment (exo-, 5 U/μl; Thermo Scientific, cat. no. EP0422) |
• dATP (100 mM; New England BioLabs, cat. no. N0440S) |
• dCTP (100 mM; New England BioLabs, cat. no. N0441S) |
• dGTP (100 mM; New England BioLabs, cat. no. N0442S) |
• T4 DNA ligase (highly concentrated; 30 Weiss U/μl; Thermo Scientific, cat. no. EL0013) |
• ATP (100 mM; Thermo Scientific, cat. no. R0441) |
• Adapters, part of the Illumina TruSeq DNA sample preparation kits (Illumina, cat. no. FC-121-2001) ▲ CRITICAL Illumina TruSeq DNA adapters are highly recommended. If they are not available, ensure that the cytosines (Cs) of the ordered oligos are methyl group-modified (mCs) and double HPLC-purified. The oligo sequenc... |
• MethyCode bisulfite conversion kit (50 reactions; Thermo Scientific, cat. no. MECOV-50) |
• tRNA (Roche, cat. no. 10109517001) |
• PfuTurbo Cx hotstart DNA polymerase (2.5 U/μl; Agilent Technologies, cat. no.600412) |
• dNTP mix (10 mM each; Clontech, cat. no. D4030RA) |
• Agencourt AMPure XP beads (Beckman Coulter; cat. no. A63881) |
may be a consequence of the low sequence complexity of scRRBS libraries. |
Data analysis. Bioinformatics analysis methods for DNA methylation profiling data of whole-genome bisulfite sequencing and RRBS have been reported previously, which are designed to map bisulfite-converted sequence reads to a reference genome and to determine cytosine methylation states at single-base resolution10,17. C... |
• Phusion high-fidelity (HF) PCR master mix with HF buffer (New England BioLabs, cat. no. M0531S) |
• Qubit dsDNA high-sensitivity (HS) assay kit (Thermo Scientific, cat. no. Q32854) |
• SYBR Gold nucleic acid gel stain (10,000×; Thermo Scientific, cat. no. S-11494) ! CAUTI0N This reagent is harmful if swallowed or inhaled, and it causes irritation to the skin, the eyes and the respiratory tract. Handle it using appropriate equipment. |
• Glycerol (Sigma-Aldrich, cat. no. G5516-100ML) |
• QIAquick PCR purification kit (Qiagen, cat. no. 28106) |
• Ethanol (Sigma-Aldrich, cat. no. E7023)! CAUTI0N Ethanol is flammable. Handle it using appropriate equipment. |
• TBE buffer (5×; Amresco, cat. no. J885-1L) |
• Ammonium persulfate (AP; Sigma-Aldrich, cat. no. A3678-100G) ! CAUTION AP is harmful if swallowed or inhaled, and it causes irritation to the skin, the eyes and the respiratory tract. Handle it using appropriate equipment. |
• N,N,N',N'-tetramethylethylenediamine (TEMED; Sigma-Aldrich, cat. no. T9281-100ML)! CAUTI0N TEMED is harmful if swallowed or inhaled, and it causes irritation to the skin, the eyes and the respiratory tract. Handle it using appropriate equipment. |
• Ammonium acetate (Amresco, cat. no. 0103-500G) |
• Magnesium acetate tetrahydrate (Amresco, cat. no. 0131-500G) |
• SDS (Amresco, cat. no. S0227-500G) |
• EDTA (Amresco, cat. no. 0322-1KG) |
• PBS buffer (pH 7.2; 1×; Gibco, cat. no. 14249-95) |
• Leukemia inhibitory factor (LIF; 10 million units/1 ml, Millipore, cat. no. ESG1107) |
• DMEM: nutrient mixture F-12 (DMEM/F-12; Gibco, cat. no. 11320-033) |
• FBS (Gibco, cat. no. 12483-020) |
• Trypsin-EDTA, 0.05% (wt/vol) (1×; Gibco, cat. no. 25300-062) |
• Albumin, acetylated from bovine serum (Ac-BSA; Sigma-Aldrich, cat. no. B8894) |
The linkage of the last two bases of the TruSeq universal adapter oligo should be phosphorothiated to avoid nuclease cleavage of the T overhang. |
The TruSeq adapter oligo (indexes 1-12) should be 5'-terminal phosphorylated for ligation of the inserted DNA fragments. |
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