Protocols: 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)
ENA first public
2022-01-01
ENA last update
2022-01-01
External Id
SAMEA9693319
INSDC center alias
Immuno-genomics & Systems Biology laboratory, Institute of Life Sciences (ILS), Bhubaneswar, Odisha, India-751023. Manipal Academy of Higher Education, Manipal,
INSDC center name
Immuno-genomics & Systems Biology laboratory, Institute of Life Sciences (ILS), Bhubaneswar, Odisha, India-751023. Manipal Academy of Higher Education, Manipal,
INSDC first public
2022-01-01T00:13:00Z
INSDC last update
2022-01-01T00:13:00Z
INSDC status
public
Submitter Id
E-MTAB-10864:Sample 5
broker name
ArrayExpress
cell line
DC1940 (MutuDC)
cell type
dendritic cell
common name
house mouse
disease
normal
organism part
spleen
phenotype
CD8alpha-positive
sample name
E-MTAB-10864:Sample 5