GSM8305569: Exp1, Vancomycin, with Candida; Mus musculus; RNA-Seq
Macrophages were seeded overnight in 24-well plates at 1x106 cells/well, and infected with C. albicans for 2 hours (MOI 2:1) or mock infected with PBS. Media was then removed from wells and 1mL of Trizol (Life Technologies) added to each well. Lysed cells were lifted from the wells, transferred to RNAse-free Eppendorfs and stored at -80°C. Samples were thawed at room temperature and 200μL of chloroform was added to each tube, mixed by inverting several times and allowed to settle for 5 minutes. Samples were centrifuged at 12,000g for 15 minutes at 4°C. The top layer containing RNA was transferred to RNAse-free Eppendorf tubes and an equal volume of 70% ethanol was added to each sample. Samples were then added to Qiagen columns (RNeasy kit, Qiagen) to be washed and perform a DNAse digest step (Qiagen). RNA was eluted in RNAse-free water, and then QC-tested using the Qubit Fluorometer TapeStation. RNA libraries were then prepared using the Lexogen QuantSeq 3' mRNA-Seq kit according to the manufacturers' instructions. Illumina sequencing was performed on the NextSeq 500 sequencing platform using the v2.5 150 cycles mid output flow cell, which produced 75 bp single reads. Reads where mapped to the Mus musculus reference genome. Read counts were quantified using HTSeq-count. Differentially expressed genes between biological conditions were detected using DESeq2 analysis pipeline in R (Padj<0.05). Lexogen 3' QuantSeq FWD