title: Sample 2
organism: Mus musculus
description: Protocols: DC1940 cell lines (mutu DCs - well characterized splenic CD8a+ DCs) were used for preparation of libraries. Stable Control and SMRT KD DC lines were generated using pre-cloned shRNA constructs under the pLK0.1 lentiviral vector backbone (Sigma). DCs were subjected with pre-incubation with modulator treatments with Mhy1485 (5μM), and DEBM (1mM) together, followed by CpG stimulation for 6h. 4 samples were prepared per replicate x 3 biological replicates, total - 12 samples. These are – 1. Control DC line CpG 6h,  2. SMRT KD DC line CpG 6h,  3. Control CpG 6h + Mhy1485 + DEBM and 4. SMRT KD CpG 6h + Mhy1485 + DEBM EDC shRNA control and SMRT shRNA KD transduced cDC1 DC line showed characteristic phenotypic and functional resemblance to type I splenic CD8α conventional DCs (PMID: 23162549). The cell line was cultured in IMDM-glutamax media supplemented with 10% FBS, Penstrep, Sodium bicarbonate, HEPES buffer, and β-mercaptoethanol, and further maintained in a humidified incubator at 37℃ with 5% CO2. For in-vitro experiments, potent activation was attained when challenged with TLR9 specific CpG-B at a concentration of 1μg/ml for 6h. DC lines were subjected with pre-incubation of modulator treatments with Mhy1485 (5μM), and DEBM (1mM) followed by CpG stimulation for 6h. Thereafter cells were harvested and subjected to RNA isolation and library preparation for NextSeq. RNA extraction was performed using a kit from Macherey-Nagel. Briefly, samples were lysed with 350ul of LBP buffer, following which genomic DNA was removed using a nucleospin gDNA removal column. The flow-through was mixed with 100ul of the binding solution, centrifuged at 11,000xg for 15 seconds, and washed using wash buffer 1 and 2 before eluting RNA in 20ul of RNAse-free water. Magnetic beads and an mRNA isolation kit were used to isolate mRNA from 1μg of total RNA (PolyA mRNA isolation module, NEB). NEB's mRNA library preparation kit Ultra II was later used to prepare RNA-seq libraries according to the manufacturer's instructions. Qubit 2.0 (Invitrogen) was used to determine library concentrations, and Tapestation was used to examine fragment sizes (Agilent). Following that, the libraries were sequenced on the Illumina NextSeq 550 platform.