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10. Transfer the supernatant to new nuclease-free tubes without disturbing the beads. |
▲ CRITICAL STEP KAPA HiFi HotStart ReadyMix can be used as an alternative to Phusion HF PCR master mix with HF buffer, if available. |
## ?TROUBLESHOOTING |
29| Add 30 μl of the second-round PCR mixture from Step 28 to the tube from Step 27 so that the total volume of the PCR mixture is 50 μl: 20 μl of eluted DNA and 30 μl of second-round PCR mixture. Mix well by vortexing and centrifuge the mixture again at 9,000g for 1 min at 4 °C. |
30| Perform another 25 PCR cycles with the following thermocycler program: |
Cycle number Denature Anneal Extend Hold |
1 98 °C, 2 min |
2-26 98 °C, 10 s 60 °C, 30 s 72 C, 1 min |
27 72 C, 5 min |
28 4℃ |
31| After the PCR cycles, centrifuge the tubes at 9,000g for 1 min at 4 °C. Next, purify the PCRs once using 1:1-fold AMPure XP beads, as described in Box 1; finally, elute in 20 μl of nuclease-free water. |
## Size selection of the amplified DNA fragments TIMING 9-10 h |
32| Prepare the 12% (wt/vol) native polyacrylamide TBE gel (Reagent Setup) using the Bio-Rad electrophoresis system. ▲ CRITICAL STEP As more primer dimers are generated in the scRRBS protocol than in regular RRBS protocols, we recommend the use of a gel-based approach to thoroughly remove the primer dimers. |
33| Add 4 μl of 6× DNA loading buffer to the sample, mix it well by vortexing and spin down the mixture briefly. |
34| Pour freshly prepared 1× TBE running buffer into the electrophoresis apparatus, and place the gel cassette into the apparatus. Then, pull out the combs carefully to avoid destroying the wells. |
35| Load 24 μl of well-mixed samples and 2 μl of low-range DNA ladder into different wells. Run the gels at 150 V for ~1 h, stopping before the bromophenol blue loading dye runs out of the gel. |
36| After electrophoresis, remove the gel cassette from the electrophoresis apparatus, peel the gel off the glass and stain it with a 1:10,000 dilution of SYBR Gold nucleic acid gel stain in 1× TBE buffer for 10 min at room temperature in darkness on a shaker. |
## PROTOCOL |
37| Wash the gel twice with 1× TBE buffer to remove the stain dye, and then visualize the gel with a Dark Reader transilluminator in darkness (Fig. 2a). |
38| Excise the gel slices containing 200-700-bp DNA fraqments using a clean disposable blade, and then transfer the gel slices into 0.5-ml thin-walled PCR tubes. |
! CAUTION Handle the blade with appropriate protection. |
39| Recover DNA fragments from the gels using an appropriate method. We have found that we achieve maximum recovery using the 'crush and soak' method (Box 2). |
40| Purify the eluate from Step 39 using the QIAquick PCR purification kit (or any equivalent kit) according to the manufacturer's protocol. Elute the DNA from the column using 50 μl of preheated EB buffer (part of the QIAquick PCR purification kit). |
41| Purify the eluate twice using 1:1-fold AMPure XP beads, as described in Box 1, and finally elute in 20 μl of nuclease-free water. |
▲ 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 |
a |
b |
C |
Figure 2 | The size distribution of the typical scRRBS libraries. (a) The polyacrylamide TBE gel results of three scRRBS libraries (before gel-based size selection); the DNA is smeared from 200 bp to ~5 kb, with some detectable adapter dimers at ~120 bp. (b,c) The Fragment Analyzer results of two scRRBS libraries estab... |
## 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. |
▲ CRITICAL STEP The standard curve-based qPCR assay is a standard quantification assessment for the Illumina libraries before deep sequencing. First, prepare serial tenfold dilutions of the standard Illumina libraries to generate the standard curve; on the basis of this curve the number of adapter-insert-adapter molecu... |
43| Assess the final libraries using a Fragment Analyzer (Advanced Analytical Technologies) to check the size distributions (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 peaks corresponding to MspI fraqments for some repetitive elements (Fig. 2b,c). If the Fraqment Analyzer results show that there are still primer dimers present, perform another clean-up step with 1:1-fold AMPure X... |
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. |
## Data analysis for single-cell RRBS data TIMING 2-3 d |
CRITICAL An overview of the major procedures involved in scRRBS data analysis is summarized in Figure 3. An archive containing custom scripts used in this protocol is available as Supplementary Data. The overview of these custom scripts, including the script names, functions and the corresponding protocol steps in whic... |
## PROTOCOL |
45| Perform quality control to filter low-quality reads and reads containing adapter sequences, as well as to remove additional bases that contain cytosines, which were artificially introduced during library preparation. This step can be conducted using the trim_galore tool with the following command line: |
trim_galore --quality 20 --phred33 --stringency 3 --gzip --length 36 --rrbs --paired --trim1 --output_dir <output_dir> <read1.fastq.gz> <read2.fastq.gz> |
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