Tissue-specific DamID protocol using nanopore sequencing

Abstract

DNA adenine methylation identification (DamID) is a powerful method to determine DNA binding profiles of proteins at a genomic scale. The method leverages the fusion between a protein of interest and the Dam methyltransferase of E. coli, which methylates proximal DNA in vivo. Here, we present an optimized procedure, which was developed for tissue-specific analyses in Caenorhabditis elegans and successfully used to footprint genes actively transcribed by RNA polymerases and to map transcription factor binding in gene regulatory regions. The present protocol details C. elegans-specific steps involved in the preparation of transgenic lines and genomic DNA samples, as well as broadly applicable steps for the DamID procedure, including the isolation of methylated DNA, the preparation of multiplexed libraries, Nanopore sequencing, and data analysis. Two distinctive features of the approach are (i) the use of an efficient recombination-based strategy to selectively analyze rare cell types and (ii) the use of Nanopore sequencing, which streamlines the process. The method allows researchers to go from genomic DNA samples to sequencing results in less than a week, while being sensitive enough to report reliable DNA footprints in cell types as rare as 2 cells per animal.

A NanoDam toolkit for tissue-specific transcription factor profiling inC. elegans

“…In the case of the RNT-1 TaDa and NanoDam comparison experiments 2 biological replicates were grown simultaneously and were collected, washed and frozen as previously described (KATSANOS AND BARKOULAS 2022). gDNA and methylated DNA was purified using a Qiagen DNeasy blood and tissue kit and processed as previously described (GOMEZ-SALDIVAR et al 2021). All subsequent steps used to purify and amplify methylated DNA by NanoDam are identical to standard C. elegans TaDa protocols.…”