Amplicon based single molecule footprinting in TC-1 DNMT TKO mESCs after RMCE - Mutant Libraries

ABSTRACT: To directly test how individual TFs combine their functions to open chromatin at specific loci, we used synthetic DNA sequences. We generated a library containing all combinations of TF-motif deletions for three CREs: a CTCF-bound region as a positive control (found in \"MutLib1\"), an enhancer with five KLF4 motifs (found in \"MutLib2\"), and a promoter with YY1, NRF1, and MYC motifs (found in \"MutLib1\"). We then inserted the library 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.  The dataset generated includes amplicon-based SMF data from the ectopic site under steady-state conditions, in mouse cells (i.e., TC-1 knock-out of the three DNA methyl transferases (DNMT TKO) mESCs). Two biological replicates were generated. 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

ORGANISM(S): Mus musculus

SUBMITTER: charles girardot 

PROVIDER: E-MTAB-16657 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress