Table 1. . Recommended epigenetic factors to be examined.

Epigenetic factor	Technique	Rationale	Feasible for low cell number approaches?
Noncoding RNA	Purification and whole transcriptome RNA sequencing (RNA-seq) adapted to different types of RNAs including miRNAs and long noncoding RNAs	Noncoding RNAs, including miRNAs and lncRNAs, are strongly implicated in gene regulation and are deregulated in disease processes. RNA-seq will simultaneously provide information on regulatory RNAs, gene expression, and splicing isoforms	Single cell RNA-seq is viable but may be very challenging for lncRNAs, many of which are lowly abundant. Single cell RNA-seq has been performed for lung cells [3] and other tissues
Open chromatin	Assay for transposase-accessible chromatin with high-throughput sequencing	This method requires fewer cells than other methods to map open chromatin, like measurement of DNase hypersensitive sites or formaldehyde-assisted identification of regulatory elements	Recently a single-cell method for ATAC-seq was published [4]
DNA methylation	Whole-genome bisulfite sequencing or nanopore-based sequencing	Genome-wide DNA methylation determination provides an unbiased assessment of the location of DNA methylation†	Single cell bisulfite sequencing has been published [5], as well as solid state nanopore-based sequencing using the methyl-binding protein MBD1 [5]
H3K4me1	ChIP-seq	A histone mark associated with active and poised enhancer regions [6]	Low-cell-number approaches include iChIP-seq [7] or recovery via protection (RP)-ChIP-seq and favored amplification RP-ChIP-seq (FARP-ChIP-seq) [8]
H3K27Ac	ChIP-seq	A histone mark associated with active enhancers and promoters [6]	As above
H3K27me3	ChIP-seq	A histone mark indicative of Polycomb repression and gene inactivity [9]	As above
CTCF	ChIP-seq	CTCF is a chromatin-organizing protein involved in looping interactions between enhancers and promoters [10], among others	Due to the lower abundance of DNA-binding factor marks, low cell assays may not yet be within reach

^†Whole-genome bisulfite sequencing does not distinguish between methyl and hydroxyl-methyl C, an oxidized version of methylcytosine that is a likely intermediate in demethylation and that is common in the nervous system [11].

RNA: seq: RNA sequencing.