alias: E-MTAB-6916:Twelve hr Control Rep2_p
title: Illumina HiSeq 2500 paired end sequencing; RNA-seq from ZEB1 Knockdown and Control CD8+cDC1
design: RNA-seq from ZEB1 Knockdown and Control CD8+cDC1
library_construction_protocol: DC Cell lines (CD8+cDC1 control and Zeb1 KD) were grown in IMDM-glutamax (GIBCO) adjusted with NaHCO3 to 310 mOsm if required and supplemented with 8–10% heat inactivated FCS (tested for endotoxin toxicity toward DC cultures), 10 mM Hepes (GIBCO 15630), 50 μM β-mercaptoethanol (GIBCO 31350), and 50 U/mL of penicillin and 50 μg/mL streptomycin (GIBCO 15070). 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 different activation media containing TLR9 agonist CpG-B at various time point 0h,6h and 12h. After stimulation, cells were preserved in RNA-later(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 2ug of total RNA was used to prepare library 2 ug of total RNA was used to isolate mRNA through magnetic beads using mRNA isolation kit (PolyA mRNA isolation Module, NEB) followed by RNA-seq library preparation using NEB Ultra RNA seq Library kit and their multiplex indexes strictly following the vendor recommended 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).
identifiers.namespace: Institute of Life Sciences
identifiers.id: E-MTAB-6916:Twelve hr Control Rep2_p