Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).
DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.
Epigenetics Programme, Babraham Institute, Cambridge, UK.
Stephen J
0000-0002-6183-491X
SJ
Clark
Epigenetics Programme, Babraham Institute, Cambridge, UK.
Sébastien A
SA
Smallwood
Epigenetics Programme, Babraham Institute, Cambridge, UK.|Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
Heather J
0000-0002-6482-0337
HJ
Lee
Bioinformatics Group, Babraham Institute, Cambridge, UK.
Felix
0000-0002-5513-3324
F
Krueger
Epigenetics Programme, Babraham Institute, Cambridge, UK.|Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.|Centre for Trophoblast Research, University of Cambridge, Cambridge, UK.|Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
Wolf
W
Reik
Epigenetics Programme, Babraham Institute, Cambridge, UK.|Centre for Trophoblast Research, University of Cambridge, Cambridge, UK.
Gavin
G
Kelsey
D000818:Animals; D001483:Base Sequence; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects*; D023281:Genomics / Q000379:methods*; D051379:Mice; D017422:Sequence Analysis, DNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D013447:Sulfites / Q000494:pharmacology*
D013447:Sulfites; C042345:hydrogen sulfite