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QP1, QP2 PCR primers Upon arrival, dissolve the forward (QP1) and reverse (QP2) primers using nuclease-free water to 100 μM as stock solutions and dilute them to 10 μM as working solutions. Both the stock and working solutions are stable for 1 year if frozen at -80 °C. |
Diffusion buffer Diffusion buffer is 500 mM ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA (pH 8.0) and 1% (wt/vol) SDS. Diffusion buffer should be stored at room temperature for no longer than 6 months. |
PBS-BSA bufferDilute Ac-BSA stock solution (20 mg/ml) with 1×PBS to 1 mg/ml as working solution and prepare aliquots in 1.5-ml Eppendorf DNA LoBind tubes. The working solution can be stored at -80 °C for at least 6 months. BSA is essential to eliminate the nonspecific binding of single cells to the surface of the react... |
Protease, 20 mg/ml Soak 20 mg of Qiagen lyophilized protease in 1 ml of 50% (vol/vol) glycerol and dissolve it for at least 30 min at 4 °C; next, prepare aliquots in 1.5-ml Eppendorf DNA LoBind tubes when fully dissolved. Store the aliquots at -20 °C for no more than 6 months. |
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 |
tRNA carrier Soak 10 mg of tRNA lyophilizate into 1 ml of nuclease-free water to allow it to dissolve for at least 10 min, and then dilute it with nuclease-free water to 10 ng/μl as the working concentration; divide the solution into 1.5-ml Eppendorf DNA LoBind tubes and store the aliquots at -80 °C for no more than 6 ... |
End-repair dNTP mix Mix and dilute the dATP, dGTP and dCTP stock solutions (100 mM each) to 1 mM, 0.1 mM and 0.1 mM, respectively. Divide the solutions into aliquots in 1.5-ml Eppendorf DNA LoBind tubes and store them at -20 °C for no more than 6 months. |
Maintenance of mESCs mESCs are maintained without feeders in the presence of LIF in DMEM containing 20% FBS for routine passage without modification. Unmethylated λ-DNA spike-in Unmethylated λ-DNA (0.3 μg/μl) should be diluted with nuclease-free water and quantified by a Qubit fluorometer and the Qubit dsDNA HS assay k... |
CT conversion reagent Add 850 μl of nuclease-free water, 50 μl of resuspension buffer (part of the MethylCode bisulfite conversion kit) and 300 μl of dilution buffer (part of the MethylCode bisulfite conversion kit) directly to the CT conversion powder (also part of the same kit). Mix the solution by brief intermittent... |
## PROCEDURE |
## Cell culture, single-cell isolation and lysis TIMING 4-5 d |
▲ CRITICAL Incubations before bisulfite conversion (i.e., before Step 18) should be performed at a temperature no higher than 80 °C; higher temperatures increase the risk of denaturing the double-stranded genomic DNA. |
▲ CRITICAL The nuclease-free water and all of the tubes used in this protocol, including the 0.2-ml thin-walled PCR tubes and the 1.5-ml Eppendorf DNA LoBind tubes, should be UV-sterilized for at least 5 min to prevent contamination. |
▲ CRITICAL All steps performed with a PCR thermocycler should be carried out on an instrument with a heated lid to avoid liquid evaporation from the reaction tubes. |
1| Aspirate the medium from mESCs maintained in 60-mm cell culture dishes (at 80% confluency) and wash the entire surface area with 1× PBS. Aspirate the PBS and add sufficient 0.05% (wt/vol) trypsin to cover the cell growth area. Return the dishes to a 37 °C incubator and incubate them for 5 min. |
▲ CRITICAL STEP One 60-mm dish at 80% confluency provides sufficient mESCs for scRRBS. |
2| After the cells have detached, add 5 ml of DMEM with 20% (vol/vol) FBS to inactivate the trypsin, gently pipette the suspension to obtain single cells and collect the cell suspension into a 15-ml Falcon conical tube. |
3| Centrifuge the mixture at 300g for 5 min at room temperature and aspirate the supernatant. |
4 Wash the cell pellet with 1 ml of PBS-BSA, centrifuge it at 300g for 5 min at room temperature and aspirate the supernatant. Repeat this step once more, and finally suspend the pellet in 20-50 μl of PBS-BSA solution. ▲ CRITICAL STEP Always use PBS-BSA instead of PBS in this step to minimize nonspecific binding to the... |
5| Prepare the cell lysis buffer as set out in the following table. Mix it well by vortexing, centrifuge the mixture at 9,000g for 1 min at 4 °C, and then divide the mixture into 0.2-ml thin-walled PCR tubes (4.8 μl in each tube). |
Component Amount per sample (μl) Final concentration |
Tris-EDTA (1 M Tris, 0.1 M EDTA) 0.1 20 mM Tris, 2 mM EDTA |
1M KCL 0.1 20 mM |
10% (vol/vol) Triton X-100 0.15 0.3% (vol/vol) |
20 mg/ml protease 0.25 1 mg/ml |
Nuclease-free water up to 4.8 |
▲ CRITICAL STEP Always include at least one 'picking-buffer-only' control when preparing the lysis buffer. |
▲ CRITICAL STEP Be sure to use protease instead of proteinase K when preparing lysis buffer, as protease can be heat-inactivated at a much lower temperature than proteinase K. |
▲ CRITICAL STEP If you are starting from single sperm cells, add 10 mM DTT to the lysis buffer to decondense the sperm nucleus. |
6] Carefully transfer single cells from Step 4 into the tubes prepared in Step 5, taking care to avoid forming bubbles. We have found mouth pipetting to be the most accurate way to achieve this40-42; however, alternative approaches include fluorescence-activated cell sorting (FACS)43, laser-assisted microdissection44or... |
▲ CRITICAL STEP The volume of carry-over PBS-BSA during mouth pipetting of single cells should be no >0.2 μl. |
▲ CRITICAL STEP It is essential to include a 'picking-buffer-only' (cell lysis buffer without cells) control to evaluate possible contamination during subsequent procedures. |
! CAUTION Be careful with the glass capillary; handle it with appropriate protection. |
! CAUTIoN Not all institutions permit mouth pipetting for manipulation of single cells, especially when working on certain types of sample (e.g., primary human tissues or some infectious samples). Alternative approaches that comply with institutional, local and governmental regulations should be adopted. |
7| Centrifuge the tubes at 9,000g for at least 1 min at 4 °C to ensure that the cell is completely seeded into the lysis buffer. ▲ CRITICAL STEP Do not vortex the mixture, as the genomic DNA is easily fragmented. |
? TROUBLESHOOTING |
8| Lyse the cells at 50 °C for 3 h, and then incubate them at 75 °C for 30 min to inactivate the protease. |
▲ CRITICAL STEP This step should be performed in a PCR thermocycler rather than in a heat block or a water bath because of its capacity for stable temperature control. |
9| Centrifuge the tubes at 9,000g for 1 min at 4 °C and immediately place the tubes on ice. |
## ? TROUBLESHOOTING |
PAUSE PoINT The lysate can be frozen at -80 °C for no longer than 3 months. However, we strongly recommend proceeding to the next step immediately. |
## MspI digestion TIMING 3-4 h |
10| Prepare the MspI digestion mixture, as described in the following table. Mix it well by vortexing, and then centrifuge the mixture at 9,000g for 1 min at 4 °C. |
Component Amount per reaction (μl) Final amount |
10 U/μl MspI 0.9 9U |
10× Tango buffer 1.8 1× |
Unmethylated λ-DNA (60 fg/μl) 1 60 fg |
Nuclease-free water up to 13 一 |
▲ CRITICAL STEP Always ensure that the λ spike-in accounts for ~1% (mass/mass) of the total genomic DNA of every picked single cell. For example, if you are starting with a single diploid mammalian cell, add 1 μl (~60 fg) of unmethylated λ-DNA to the digestion mixture. |
11| Add 13 μl of MspI digestion mixture from Step 10 to each tube from Step 9 so that the total volume of each sample is 18 μl: 4.8 μl of lysis buffer, 0.2 μl of carry-over PBS-BSA (single cell included) and 13 μl of MspI digestion mixture. Mix well by vortexing the tubes, and centrifuge them at 9,000g for 1 min at 4 °... |
12| Incubate the reaction at 37 °C for 3 h, and then at 80 °C for 20 min to inactivate the restriction enzyme. Centrifuge the tubes at 9,000g for 1 min at 4 °C, and place the tubes on ice immediately. Proceed to the next step immediately. |
## End-repair/dA-tailing reaction TIMING 1-2 h |
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