Single-Cell 5fC Sequencing
Active DNA demethylation plays important roles in the epigenetic reprogramming of developmental processes. 5-formylcytosine (5fC) is produced during active demethylation of 5-methylcytosine (5mC). Here, we describe a technique called CLEVER-seq (Chemical-
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Introduction
1.1 Methods to Analyze 5-Formylcytosine
5-Formylcytosine (5fC) is produced during active demethylation of 5-methylcytosine (5mC): 5mC is sequentially oxidized by ten-eleven translocation (TET) family proteins to give 5-hydroxymethylcytosine (5hmC), 5fC and 5-carboxylcytosine (5caC); the latter two can be reversed to cytosine by thymine DNA glycosylase (TDG)-mediated DNA base-excision repair [1–6]. Active DNA demethylation is shown to have has crucial roles in multiple biological processes, including embryo development, neurogenesis, carcinogenesis, and stem cell pluripotency and differentiation [7–9]. Unlike 5mC and 5hmC, 5fC could not be distinguished from 5caC and unmodified cytosine during bisulfite treatment [10]. Various modified bisulfite-dependent methods have been developed to profile 5fC at single-base resolution [11]. fCAB-seq (chemicalassisted bisulfite sequencing for 5fC) uses EtONH2 to react specifically with 5fC and makes it resistant to bisulfite treatment. After bisulfite conversion, 5fC could be identified by subtracting traditional BS-seq signal [12, 13]. redBS-seq (reduced bisulfite sequencing) converts 5fC to 5hmC by NaBH4 treatment; thus, the converted 5fC stays intact in the bisulfite treatment, so 5fC signal
Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_16, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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could be resolved by subtracting traditional BS-seq signal [14]. In MAB-seq (M.SssI methylase-assisted bisulfite sequencing), M.SssI was used to convert unmodified cytosine (C) into 5mC in a CpG context in vitro and the methylated DNA was then subjected to bisulfite treatment. Hence, 5fC and 5caC could be indiscriminately identified [15–18]. Meanwhile, bisulfite-free 5fC profiling methods have also been developed: 5fC-targeted antibody-based [19] or chemical labeling-based [12, 20, 21] enrichment of target DNA for sequencing, and converting 5fC to 5hmC then using 5hmCsentitive restriction enzyme for detection [22]. We have also developed a bisulfite-free, single-base resolution 5fC sequencing method, fC-CET [23]. However, these methods are all limited to analysis of bulk samples. scMAB-seq and liMAB-seq could be adopted for single cell or limited cells [24] as start material but identify 5fC/5caC indiscriminately. In this chapter, we provide a detailed description of “CLEVER-seq,” a single-base and single-cell resolution 5fC mapping technology that we recently developed [25]. 1.2 Principle and Application of CLEVERseq
CLEVER-seq was developed based on the reactivity of malononitrile to 5fC (Fig. 1a, b) [25]. Malononitrile is a stable and commercially available small molecule and is highly water-soluble. Singlecells are lysed in individual tubes and labeled with malononitrile. This labeling reaction is performed under mild condition and causes no DNA degradation. After chemical treatment, the 5fC-adduct (“5fC-M”)
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