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* Phusion high-fidelity PCR master mix or KAPA HiFi HotStart ReadyMix |
Molecular event: |
A second PCR round further enriches the scRRBS library. |
Product structure: |
* amplified scRRBS library molecules |
Step 8. Size selection and sequencing |
Input substrate: |
* amplified scRRBS library molecules |
Added oligos/reagents: |
* native polyacrylamide TBE gel |
* AMPure XP beads |
Molecular event: |
Amplified DNA fragments are size-selected, typically keeping fragments in the 200–700 bp range. The final library is quantified, quality controlled, and sequenced as paired-end Illumina libraries. |
Product structure: |
* final size-selected scRRBS sequencing library |
Final canonical library structure |
Simplified segment-level structure: |
TruSeq universal adapter + bisulfite-converted MspI genomic insert + indexed TruSeq adapter |
Source-visible sequence-level structure with generic genomic insert: |
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T |
+ bisulfite-converted MspI genomic insert |
+ GATCGGAAGAGCACACGTCTGAACTCCAGTCAC |
+ 6-bp sample index |
+ ATCTCGTATGCCGTCTTCTGCTTG |
Sequencing read interpretation: |
* Read 1 and Read 2 sequence the bisulfite-converted reduced-representation genomic insert from opposite adapter ends. |
* The indexed TruSeq adapter provides a 6-bp sample index. |
* The library is used for DNA methylation profiling after bisulfite-aware alignment and methylation calling. |
Human-curation notes |
1. The protocol table explicitly lists the TruSeq universal adapter and TruSeq indexed adapters 1–12. All adapter sequences are written 5' to 3'. |
2. The indexed TruSeq adapters contain 6-base sample indexes. |
3. The protocol notes that the last two bases of the TruSeq universal adapter should be phosphorothioated to avoid nuclease cleavage of the T overhang. |
4. The protocol notes that TruSeq indexed adapters 1–12 should be 5'-terminal phosphorylated for ligation to inserted DNA fragments. |
5. QP1 and QP2 primer sequences are explicitly printed in the protocol materials section and should be included in the sequence list. |
6. The one-tube reaction integrates cell lysis, MspI digestion, end repair/dA-tailing, adapter ligation, and bisulfite conversion before DNA purification. |
7. The final genomic insert should be interpreted as a bisulfite-converted MspI genomic insert, not ordinary unconverted gDNA. |
8. The exact genomic insert sequence is sample dependent and should not be fixed. |
# Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing |
Hongshan Guo1,5, Ping Zhu1,2,5, Fan Guo1, Xianlong Li1, Xinglong Wu1,2, Xiaoying Fan1, Lu Wen1,3,4 & Fuchou Tang1,3,4 |
1Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, Beijing, China. 2Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China. 3Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking University, Beijing, China. 4Ce... |
Published online 2 April 2015; doi:10.1038/nprot.2015.039 |
The heterogeneity of DNA methylation within a population of cells necessitates DNA methylome profiling at single-cell resolution. Recently, we developed a single-cell reduced-representation bisulfite sequencing (scRRBS) technique in which we modified the original RRBS method by integrating all the experimental steps be... |
## INTRODUCTION |
DNA methylation of cytosine is a well-known epigenetic modification that is involved in gene expression regulation¹. In mammals, DNA methylation is relatively stable in differentiated cells; by contrast, global demethylation and remethylation occur during early embryonic development and primordial germ cell development... |
## Development of the procedure |
We recently developed scRRBS for profiling DNA methylation at the single-cell level10,17. In a typical mammalian cell, there are only two copies of DNA molecules, and each comes from one of the two parents. If aiming to profile the DNA methylome of a single cell, it is crucial to avoid DNA loss as far as possible, as a... |
When starting with a single mouse diploid cell, scRRBS covers, on average, 1 million CpG dinucleotides; this accounts for ~40% of the CpG sites that can be recovered by standard RRBS using thousands of cells (on average, 2.5 million CpG dinucleotides). Importantly, ~70% of CGIs in the mouse genome can be captured. The ... |
## Overview of the procedure |
The scRRBS approach starts with cell picking and lysis in 5 μl of lysis buffer, which contains protease to release the genomic DNA. The next three steps, including the MspI digestion, the end repair/dA tailing, and the adapter ligation, are accomplished by adding the corresponding reaction components sequentially, and ... |
Figure 1 | Flowchart of the experimental procedures of the scRRBS technique. Notably, we integrated cell lysis, MspI digestion, end repair/dA tailing, adapter ligation and bisulfite treatment into a single-tube reaction to avoid unnecessary DNA loss. |
Comparison of scRRBS and single-cell bisulfite sequencing Recently, Smallwood et al.32reported an alternative scBS technique. The authors used the single-tube reaction strategy to improve a previous protocol that was based on bisulfite conversion followed by random priming33. When starting with a single mouse diploid c... |
## Applications of scRRBS |
The advantage of the scRRBS method is its applicability to subnanogram levels of DNA as starting material, down to a single cell. This is particularly useful when the starting materials are very limited and precious, such as mammalian early embryos and primordial germ cells. It also enables the heterogeneity of DNA met... |
## Limitations of single-cell methylation profiling techniques |
The first limitation of the scRRBS technique stems from the design of the RRBS method; that is, although it captures a large portion of CGIs and promoters, it provides representative, but lower, coverage of CpG-sparse regions such as enhancers27. It should be noted that the scBS technique is also biased toward CpG-rich... |
## Experimental design |
Starting material. We have successfully applied this protocol to a variety of mammalian cells, including human and mouse embryonic stem cells (mESCs), oocytes, sperm, early preimplantation blastomeres and cancer cells. The method is likely to be applicable to most mouse and human cell types, regardless of whether they ... |
Controls. It is important to avoid any DNA contamination in scRRBS experiments. All reagents and consumables used in this protocol should be free of exogenous DNA, and picking-bufferonly' controls (i.e., only pick the PBS-BSA buffer but no cells into the lysis buffer) are always essential for evaluating possible contam... |
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