The cutaneous response to a mosquito bite at different diurnal time point
The cutaneous response to a mosquito bite at different diurnal time point
The mosquito bite sites were then marked with surgical skin marker. 24 hours later, 3.5mm ear punch biopsies were taken. Tissues are collected into RLT buffer (Qiagen Cat#79216) and immediately subjected to RNA extraction as described below. RNA extraction was performed from murine skin using the RNeasy fibrous tissue mini kit (Qiagen 74704) following the manufacturer's protocol. Briefly, skin tissues were homogenized using a KIMBLE pestle inside a centrifuge tube for 30s-1min or until most tissues were lysed. Proteinase K was then added, and tissues were incubated in a 55C water bath for 10min. The mixture was spun at 10,000g for 5min and supernatant collected in a new tube. Ethanol was added to the supernatant, gently pipetted for mixing, and transferred to an RNeasy Mini column. Columns were spun to allow RNA to bind to the silica, washed with RW1, RDD, and RPE buffer per manufacturer's protocol. Samples were eluted with 30ul RNAse-free water and subjected to RNA-sequencing.  RNA libraries were prepared by removal of ribosomal RNA