Chromosome conformation analysis of N1E115 cells by HiC

Neuronal nitric oxide synthase 1 (NOS1) produces the gaseous signaling molecule nitric oxide (NO), which plays important roles in the development and function of the nervous system. The regulation of Nos1 gene expression is incompletely understood. Here, we explored the role of physiological hypoxia in the control of Nos1 transcription and the underlying mechanisms using N1E115 mouse neuroblastoma cells as a model. N1E115 cells were cultured for 3 days at high (20%) and low (4%) oxygen levels. Following the verification of upregulation of Nos1 mRNA and protein levels in response to 4% oxygen, we analyzed 3-dimensional chromosome conformation by HiC. N1E115 cells were cultured in the presence of 20% or 4% oxygen (at ambient barometric pressure). Cells were cross-linked with 1% formaldehyde and quenched by 2.5 mol/L glycine and processed using the Arima, Inc. HiC kit (Cat# A510008) or using NcoI followed by established protocol. Sequencing libraries were prepared with the KAPA Hyper Prep kit (Roche, Cat# KK8500) and sequenced using an Illumina HiSeq 4000 instrument.