GSM5353211: RNAseq_whole_ear_skin_Sepi_aCTLA4_rep2; Mus musculus; RNA-Seq
Mice were sacrificed 8 days after the first topical association of S. epi. Single-cell suspension from the ear pinnae were isolated as previously described were digested in RPMI 1640 containing 2 mM L-glutamine, 1 mM sodium pyruvate and nonessential amino acids, 20 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mM β-mercaptoethanol, and 0.25 mg/ml Liberase purified enzyme blend (Roche Diagnostic Corp.) and incubated for 1 hour 45 minutes at 37°C in 5% CO2. Digested skin sheets were homogenized using the Medicon/Medimachine tissue homogenizer system (Becton Dickinson). A ~1-mm skin region was micro-dissected at Day 8, submerged in RNAlater (Sigma Aldrich) and stored at −20°C. Total tissue RNA was isolated from skin tissue using the RNeasy Fibrous Tissue Mini kit (Qiagen), as per manufacturer's instructions. Total mRNA library was prepared using 500 ng of input RNA with the Universal Plus# RNA-seq with NuQuant Library Prep Kit (Tecan) as per manufacturer's instructions. Libraries were quantified using an Agilent Tapestation (High Sensitivity D1000 ScreenTape) and Qubit (Thermo Fisher Scientific). Libraries were pooled at equimolar concentrations and sequenced on an Illumina Nextseq 550 using the High Output v2 kit (75 cycles). Data was demultiplexed with blc2fastq. 