Sample 1
Mus musculus
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.
ENA-FIRST-PUBLIC
2024-01-01T01:01:31Z
ENA-LAST-UPDATE
2024-01-01T01:01:31Z
External Id
SAMEA110586145
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
2024-01-01T01:01:31Z
INSDC last update
2024-01-01T01:01:31Z
INSDC status
public
Submitter Id
E-MTAB-12066:Sample 1
broker name
ArrayExpress
cell_line
MutuDC1940
cell_type
CD8alpha-positive conventional dendritic cell
common name
house mouse
disease
normal
genotype
pLK0.1 empty vector
immunophenotype
CD11chigh, B220−, DEC205+, CD24high, CD11b−
isolate
not applicable
organism part
spleen
sample name
E-MTAB-12066:Sample 1
scientific_name
Mus musculus
strain
CD11c:SV40LgT-transgenic C57BL/6