Profiling chromatin accessibility in Enteric Nervous System progenitors during neurogenic phases

We utilized a Phox2b-H2BCerulean transgene that is expressed at low levels in Enteric Nervous System progenitors (ENPs) and enteric glia that is also expressed at notably higher levels in differentiating enteric neurons to capture these populations during neurogenic phases of ENS development. Collected tissues from 16.5 days post coitus developing mouse intestine included the stomach, small intestine, and colon that were dissociated to single cell suspensions for flow sort capture of Phox2b-H2BCerulaen+ cells. We applied differential gating in flow sorts to capture populations expressing low levels of the Phox2b-H2BCerulaen transgene (ENPs and enteric glia) and while concurrently collecting cells expressing high levels of the Phox2b-H2BCerulaen transgene (developing and maturing enteric neurons) from the same samples. Nuclei were generated from these Phox2b-H2BCerulaen “high” and “low” populations and encapsulated separately to produce single nucleus ATAC-Seq libraries using 10X Genomics chemistry. Sequencing was performed on the Illumina NovaSeq6000 (S4) using PE150 Sequencing targeting >50,000 reads per nucleus. Comparison of the resulting sequence data from the “high” versus “low” cell populations allowed us to identify differentially accessible genome regions in developing enteric neurons compared to ENPs and enteric glia. Overall design: 16.5 days post coitus (dpc) embryonic fetal guts derived from C3Fe.B6 Phox2b H2B-CFP transgenic lines were isolated and dissociated via cold-active protease, then flow sorted for low intensity CFP+ and high intensity CFP+ separately in two samples. Nuclei were isolated from viable, CFP+ cells. 10x Genomics' snATAC-seq platform was used for encapsulation and Illumina next-generation sequencing was performed to generate datasets. CellRanger was used to align FASTQ files to mm10. Two "dim" samples and one "bright" sample were processed further, while the other "bright" sample (4828-MS-16) was excluded due to quality concerns.