Amplicon based single molecule footprinting in TC-1 DNMT TKO mESCs after RMCE and after 24h p300 inhibition

ABSTRACT: To quantify the contribution of specific transcription factor binding versus chromatin context in chromatin opening, we inserted libraries containing hundreds of CREs into a landing pad within a neutral chromatin environment, devoid of activating or repressive chromatin modifications, in mESCs using Recombination-Mediated Cassette Exchange (RMCE). Chromatin accessibility of the inserted fragments was then profiled by targeted SMF using PCR primers that anneal to a synthetic flanking sequence, allowing unambiguous differentiation of the ectopic site from its endogenous counterpart. To investigate whether H3K27Ac contributes to chromatin accessibility at enhancers, we globally reduced H3K27Ac levels by chemically inhibiting the histone acetyltransferase p300 with the small molecule A-485 (24 hours treatment, final concentration: 3 μM). This was performed in the same mESCs carrying the CREs at the landing pad. The dataset generated includes amplicon-based SMF data from the ectopic site in DMSO- and A-485-treated conditions in mouse cells (i.e., TC-1 knock-out of the three DNA methyl transferases (DNMT TKO) mESCs). Two biological replicates were generated for each condition. In summary, cells were collected for SMF, which marks accessible cytosines via recombinant methyltransferases, followed by bisulfite sequencing to infer protein-DNA interactions and chromatin accessibility at single-molecule resolution. The sequencing library was prepared using the NEBNext DNA Ultra II Library Prep Kit and sequenced on an Illumina platform, using either a MiSeq 250 bp paired-end run, a MiSeq i100 250 bp paired-end run, or a NextSeq 2000 P1 300 bp paired-end run. Reads were pre-processed with TrimGalore and a custom R script was used to trim the plasmid backbone from the reads. After, pre-processed reads were aligned using QuasR. Further analyses were conducted using custom scripts available at https://github.com/Krebslabrep/TF-chromatin.git.

INSTRUMENT(S): Illumina MiSeq, NextSeq 2000

ORGANISM(S): Mus musculus

SUBMITTER: charles girardot 

PROVIDER: E-MTAB-16659 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress