GSM6690522: TCDD CD44hiCD8+_2; Mus musculus; RNA-Seq
Mediastinal lymph nodes (MLNs) were harvested from female offspring on day 9 post-IAV infection. Single cell suspensions of MLN cells were stained with fluorochrome-conjugated antibodies against CD3, CD8, and CD44, then immediately sorted into CD3negCD4negCD8+CD44lo and CD3negCD4negCD8+CD44hi fractions using a BD FACS Aria II cell sorter (San Jose, CA). One ng of total RNA was pre-amplified with the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech, Mountain View, CA) per manufacturer's recommendations. The quantity and quality of the subsequent cDNA was determined using the Qubit Fluorometer (Life Technologies, Carlsbad, CA) and the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). cDNA (1 ng) 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 100 nucleotides were generated for each sample using Illumina's HiSeq2500v4.