Regulation of a novel lncRNA irf8 by the Ikzf1 Myb complex drives neutrophil development

Tg(coro1a:DsRed) embryos from WT and ikzf1D4+3/D4+3; mybhkz3/hkz3 (DM) embryos at 30 hours post fertilization (hpf) were collected and dissociated using a 1 U mL-1 Papain Dissociation Kit (Worthington) in Dulbeccos phosphate buffered saline (DPBS) at 37 C for 20-30 minutes. Single cell suspensions were generated by gentle trituration, passed through a 40 um cell strainer, and subjected to fluorescence activated cell sorting (FACS). The sorted cells were subsequently processed for Smart seq. Tg(coro1a:DsRed) embryos from WT, Tg(coro1a:HA-ikzf1), Tg(coro1a:FLAG-myb), ikzf1+/+; myb+/+ (WT), and ikzf1D4+3/D4+3; mybhkz3/hkz3 (DM) embryos at 30 hpf were collected and dissociated using a 1 U mL-1 Papain Dissociation Kit (Worthington) in DPBS at 37 C for 20-30 minutes. Single cell suspensions were generated by gentle trituration, passed through a 40 um cell strainer, and subjected to FACS. Approximately 5000 sorted cells were subsequently processed for ATAC seq. Single cell suspensions of FACS sorted coro1a-DsRed cells from more than 30 embryos at 24 hpf were resuspended in DPBS as described in a previous study. The cell suspensions were filtered through a 70 um cell strainer before sorting. The collected coro1a-DsRed positive cells were loaded onto the channels of Single Cell G Chip (v3.1 chemistry, PN1000120).