Single-cell 5-hydroxymethyl cytosine dynamics during remote memory formation

We introduce the Split-Pool based Epigenetic Decoding (SPEED) method, a combinatorial barcoding strategy enhancing the throughput of single-cell 5-hydroxymethyl cytosine (5hmC) or 5-methylcytosine (5mC) profiling. SPEED enables simultaneous, unbiased examination of 5hmC traits across distinct brain regions, surpassing limitations of individual cell scrutiny. Using SPEED we unveil dynamic 5hmC patterns linked to remote memory formation in the brain. Overall design: We isolated and crosslinked nuclei, fragmented and end-repaired DNA, performed three rounds of barcode ligation within nuclei, extracted genomic DNA, biotin-labeled 5-hydroxymethylcytosine (5hmC), and enriched 5hmC-containing fragments using Streptavidin beads. Subsequently, we eluted the 5hmC-containing fragments, and libraries were constructed for further analysis.