title: GSM5425722: L1_CD49a; Mus musculus; RNA-Seq
library_construction_protocol: Intravenous labelling was conducted with anti-CD45 antibody before lung harvesting. After harvest and a single cell suspension was obtained, cells were either restimulated with anti-CD2/CD28 or not restimulated, then sorted into four groups based off of integrin expression. All cells were live, singlet, CD44+CD8+ cells. Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Valencia, CA) per manufacturer's recommendations. 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). 1ng of total RNA was pre-amplified with the SMARTer Ultra Low Input kit v4 (Clontech, Mountain View, CA) per manufacturer's recommendations. The quantity and quality of the subsequent cDNA was determined using the Qubit Flourometer (Life Technnologies, Carlsbad, CA) and the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). 150pg 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). Single end reads of 100nt were generated by the HiSeq2500 (Illumina, San Diego, CA).