NextSeq 550 paired end sequencing; RNA-seq of Control and SMRT KD CD8α+ DCs at 0hr and 6hr CpG stimulation
RNA-seq of Control and SMRT KD CD8α+ DCs at 0hr and 6hr CpG stimulation
DC Cell lines (CD8+cDC1 control and SMRT KD) were grown in IMDM-glutamax (GIBCO) adjusted with NaHCO3 supplemented with 10% heat inactivated FCS, 10 mM Hepes, 50 μM β-mercaptoethanol, and 50 U/mL of penicillin and 50 μg/mL streptomycin. The cells were maintained at 37°C in a humidified incubator with 5% CO2. These DCs were dissociated with short incubation in non-enzymatic, 5 mM EDTA-based cell dissociation buffer (5 mM EDTA in 20 mM Hepes-PBS). DCs were plated in 6-well plates at a density of 5x105 cells/ml overnight. The cells were then challenged with activation media containing TLR9 agonist CpG-B at 0hr and 6hr. After stimulation, cells were preserved in lysis buffer (LBP lysis buffer, Macherey-Nagel) and -80oC until further RNA isolation and processing of samples. The cells preserved in LBP lysis buffer were first taken out from -80°C and thawed by placing the plates/tubes on ice. Total RNA was isolated using NucleoSpin RNA Plus kit (Macherey-Nagel) according to manufacturer's protocol. RNA concentration was estimated by nanodrop (Thermo) and then 500ng of total RNA was used to prepare library. mRNA from samples were isolated using magnetic beads from mRNA isolation kit (PolyA mRNA isolation Module, NEB) followed by RNA-seq library preparation using NEB Ultra II RNA seq Library kit and their multiplex indexes were strictly followed based on the recommendation in the protocol. After library preparation concentration of libraries were estimated using qubit 2.0 (Invitrogen) and the recommended fragmentation sizes were confirmed by Bio-analyzer (Agilent)