title: GSM4276346: 5: Vehicle-Naive2_RNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Peripheral lymph nodes (cervical, axillary, brachial, inguinal, and sacral) were harvested from naïve adult developmentally exposed mice. Mediastinal lymph nodes were harvested from adult developmentally exposed mice on day 9 post IAV infection. Lymph nodes were processed to single cell suspensions, and CD4+ T cells were purified to >90% purity using the MojoSort Mouse CD4+ T cell negative selection isolation kit protocol (BioLegend, San Diego, CA). RNA was isolated from total CD4+ T cells using the RNeasy Mini kit (Qiagen, Valencia, CA). Total RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). RNA (1 ng) was pre-amplified with the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech, Mountain View, CA) following the manufacturer's recommendations. The quantity and quality of the subsequent cDNA was determined using the Qubit Fluorometer (Life Technnologies, Carlsbad, CA) and the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). For total CD4+ T cells, 150 pg of cDNA was used to generate Illumina compatible sequencing libraries with the NexteraXT library preparation kit (Illumina, San Diego, CA) per manufacturer's protocols. The amplified libraries were hybridized to the Illumina single end flow cell, and amplified using the cBot (Illumina, San Diego, CA) at a concentration of 10 pM per lane. Single end reads of 100nt were generated for each sample using Illumina's HiSeq2500v4.
attributes.tag: GEO Accession
attributes.value: GSM4276346