Illumina HiSeq 4000 paired end sequencing; RNA-seq of cells in nasal turbinates with or without 7DW8-5 treatment
The skin at the nape of the neck was incised with fine dissecting scissors, and the incision rotated around the entire neck. This skin was then dissected anteriorly and completely removed, exposing the bone over the entire skull and nose. The skull was then sectioned in the coronal plane posterior to the eyes. The remnant of the brain was completely removed, leaving the anterior aspect of the skull base exposed. The most anterior point of the skull base on either side is directly posterior to the mouse nasal cavities. The remaining portion of the skull was removed to the posterior aspect of the nasal cavity. A scissor was inserted into the posterior aspect of the nasal cavity, and the suture line was incised separating one side of the septum (medially) from the maxilla (laterally). The scissor was then turned inferiorly to section the palate, and thus completely separated the septum from the lateral nasal wall. The procedure was then repeated on the other side. The remaining attachment at the nasal tip was severed and the septum completely removed. Total RNA was extracted with miRNeasy micro kit (Qiagen, 217084) according to the manufacturer's instructions. ERCC RNA Spike-In Mix kit (ThermoFisher Scientific, 4456740) was added to normalized total RNA prior to library preparation following manufacturer's protocol. The RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina according to manufacturer's instructions (New England Biolabs, Ipswich, MA, USA).