title: Illumina HiSeq 2000 sequencing; RNAseq of skin vaccination array
design: RNAseq of skin vaccination array
library_construction_protocol: Ear skin samples were obtained 4 hours post vaccination and was snapped frozen using liquid nitrogen. All biopsies were weighted and kept at -80 °C for further RNA-seq analysis.  ID injections were prepared by diluting Intanza ®2013 in dPBS to the appropriate dose (30 ng) and administered to the ventral side of murine right ears.  To induce physical immune enhancements while vaccinating into the skin, the Nanopatch™ was used. These Nanopatch™ were manufactured as described elsewhere, measuring 4 by 4 mm with a density of 20,000 microprojections per cm2, and microprojections were 110 µm long. The Nanopatch™ were cleaned with 70% ethanol, rinsed with MilliQ water and air dried. The Nanopatch™ vaccine formulation consisted of 1% methylcellulose (Methocel 60 HG; Fluka/Sigma-Aldrich), Intanza®2013 and dPBS as the diluent. Vaccine formulation was dry coated on to the Nanopatch™ by a 70° and 20° angle nitrogen gas jet. One patch per mouse was used to vaccinate mice with a spring-loaded applicator at a constant velocity to penetrate microprojections into the ear skin of mice, left for 2 minutes to allow the reconstitution and diffusion of the dry coated vaccine. Ear skin samples were harvested and stored at -80°C until being processed. TissueRuptor® (9001274; QIAGEN) was used to disrupt and homogenise skin in 1 ml of QIAzol Lysis Reagent. Total RNA were extracted using QIAGEN RNeasy® Microarray Tissue Kit (73304; QIAGEN) with an additional genomic DNA removal step (RNase-free DNAse set; 79254; QIAGEN) according to manufacturer's protocol.  Complementary DNA libraries were made using TruSeq RNA Sample Preparation kit (RS122-2001/2; Illumina)
attributes.tag: Experimental Factor: study group
attributes.value: IntradermalNeedleSyringe_vaccine