BioProject

Purpose: The goal of this study is to identify the differential cardiac chromatin accessibility between WT and cardiomyocyte conditional knockout (Chd4-CMko) hearts at E10. More...

Purpose: The goal of this study is to identify the differential cardiac chromatin accessibility between WT and cardiomyocyte conditional knockout (Chd4-CMko) hearts at E10.5 using ATAC-seq. Methods: Three hearts at E10.5 were pooled per genotype per replicate, and were then dissociated into single cells. 40,000 viable cells were taken for were lysed to isolate nuclei, which were treated with Tn5 transposase (Nextera DNA Sample Prep Kit, Illumina) to isolate DNA. Fragmented DNA was then amplified using bar-coded PCR primers and libraries were seuqenced. Results: 15736 differential peaks (2-fold change) were identified between E10.5 WT and Chd4-CMko hearts. Overall design: Each embryonic mouse heart was dissected in cold PBS and individually cryopreserved in 200 mL of DMEM medium with 10% FBS and 10% DMSO (vol/vol) using a Mr. Frosty isopropyl alcohol chamber. Hearts from each genotype were thawed in a 37℃-water bath and pooled (three pooled E10.5 hearts per genotype per biological replicate, four biological replicates for each genotype). Samples were pelleted at 1000g for 3 min at 4℃. The medium was aspirated, and the hearts were washed with cold PBS. Hearts were then dissociated in 200 mL of 0.05% Trypsin/EDTA at 37℃ for 10 min, with gentle agitation every 3 min. Dissociation was stopped by adding 400 mL of DMEM/10% FBS (vol/vol), and the samples were passed through a 100-micron cell strainer to remove connective tissue. Cells were quantified using Trypan blue, and 40,000 viable cells were taken for downstream procedures. Dissociated embryonic cardiac cells were lysed to isolate nuclei, which were treated with Tn5 transposase (Nextera DNA Sample Prep Kit, Illumina) to isolate DNA Fragmented DNA was then amplified using bar-coded PCR primers and libraries were pooled. Pooled libraries were then sequenced with the NovaSeq 6000 platform. Reads were aligned to the mm10 reference genomes using BWA-MEM and peaks were called using HOMERv4.10.3. Annotation and analysis were performed using ChIP-Seeker v1.20.0. Less...