characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2024-01-01T01:01:31Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2024-01-01T01:01:31Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA110586155
characteristics.characteristic: External Id
characteristics.text: Immuno-genomics & Systems Biology laboratory, Institute of Life Sciences (ILS), Bhubaneswar, Odisha, India-751023. Manipal Academy of Higher Education, Manipal,
characteristics.characteristic: INSDC center name
characteristics.text: 2024-01-01T01:01:31Z
characteristics.characteristic: INSDC first public
characteristics.text: 2024-01-01T01:01:31Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS12683777
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12066:Sample 5
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: MutuDC1940
characteristics.characteristic: cell_line
characteristics.text: CD8alpha-positive conventional dendritic cell
characteristics.characteristic: cell_type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: DC1940 cell lines (mutu DCs - well characterized splenic CD8a+ DCs) were used for the preparation of libraries. Stable Control (EDC) and NCoR1 KD DC lines were generated using pre-cloned shRNA constructs under the pLK0.1 lentiviral vector backbone (Sigma). DCs were subjected with 1h pre-incubation with inhibitor treatments with KC7F2 (10μM), and etomoxir (50μM) together, followed by CpG stimulation for 6h. Three indendent biological replicate were generated for each condition given below. 1. Control DC line CpG 6h, 2. NCoR1 KD DC line CpG 6h, 3. Control CpG 6h + KC7F2 + Etomoxir and4. NCoR1 KD CpG 6h + KC7F2 + Etomoxir EDC shRNA control and NCoR1 shRNA KD transduced cDC1 DC line was gifted by Prof. Hans Acha-Orbea. The DC line showed characteristic phenotypic and functional resemblance to type I splenic CD8α conventional DCs (PMID: 23162549). The cell line was cultured in IMDM-glutamic 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. EDC and NCoR1 KD DC lines were subjected to 1h pre-incubation of inhibitor treatments with KC7F2 (10μM), and etomoxir (50μM) followed by CpG stimulation for 6h. Thereafter cells were harvested and subjected to RNA isolation and library preparation. 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 Bio-analyzer was used to examine fragment sizes (Agilent). Following that, the libraries were sequenced on the Illumina NextSeq 550 platform.
characteristics.characteristic: description
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: pLK0.1-NCoR1 knockdown
characteristics.characteristic: genotype
characteristics.text: CD11chigh, B220−, DEC205+, CD24high, CD11b−
characteristics.characteristic: immunophenotype
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: spleen
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: CD11c:SV40LgT-transgenic C57BL/6
characteristics.characteristic: strain
characteristics.text: Sample 5
characteristics.characteristic: title