NextSeq 550 paired end sequencing; RNA-seq of Control and NCoR1 KD CD8α+ DCs at 0hr and 6hr of stimulation
RNA-seq of Control and NCoR1 KD CD8α+ DCs at 0hr and 6hr of stimulation
DC Cell lines (CD8+ cDC1 control and NCoR1 KD) were grown in IMDM medium supplemented with 10% FBS and antibiotic solution, 5 x 10-5 M b-mercaptoethanol, sodium bicarbonate and HEPES. The cells were maintained at 37°C in a humidified incubator with 5% CO2. These DCs were dissociated with a short incubation of 2-3 min in a nonenzymatic, 5 mM EDTA in 20 mM HEPES buffer. Cells were plated in 6-well plates at a density of 5 x 105 cells/ml overnight. The cells were then challenged with different activation media containing IFNg, TLR3 agonist pIC, and CpG+pIC+IFNg for 2hr or 6hr. For performing RT-qPCR analysis the cells were washed in the plate once with PBS followed by addition of RNA-later (RLT) buffer (Qiagen) for lysis of cells. The plates were then stored at -80oC until further RNA isolation and processing of samples. mRNA was isolated from 2ug of total RNA using magnetic beads mRNA isolation kit (PolyA mRNA isolation Module, NEB) RNA-seq library preparation was performed using mRNA library preparation kit (NEB)