Mus musculus
Protocols: For sampling cell culture medium was removed and to lyse cells 1000 mikroliter phenol/guanidinium thiocyanate solution (peqGOLD TriFast(TM), VWR) was added to the cell layer. Cells were then passed several times through a pipette. Cells were incubated in cell culture medium (alpha-MEM, 100 U/ml Penicillin, 100 mikrogram/ml Streptomycin, 10 % heat-inactivated fetal calf serum and 568 mikromolar L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate), with DMSO in final concentration of 0.02%. Mesenchymal stem cells (MSCs) were cultured in alpha-MEM, 100 U/ml Penicillin, 100 mikrogram/ml Streptomycin and 10 % heat-inactivated fetal calf serum. For assays, cells were plated at a density of 3500 cells per cm² in 35 mm cell culture dishes. Upon reaching 100 % confluency medium was changed to alpha-MEM, 100 U/ml Penicillin, 100 mikrogram/ml Streptomycin, 10 % heat-inactivated fetal calf serum and 568 mikromolar L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate. Medium contained vehicle (DMSO), tofacitinib (500 nM) or baricitinib (300 nM), with DMSO being adjusted to 0.02% in all samples. TriFast-Cell-Lysates were incubated for 5 minutes at room temperature. 200 mikroliter chloroform was added and samples shaken vigorously by hand for 15 seconds. After incubation at room temperature for 10 minutes, samples were centrifuged (4°C, 5 minutes, 12000 xg). The uppermost colorless aqueous phase was transferred into a fresh reaction tube and the RNA herein was precipitated with 500 mikroliter isopropanol on ice for 15 minutes. RNA was pelleted by centrifugation (4°C, 10 minutes, 12000 xg). Supernatant was discarded and RNA pellet washed twice with 75% ethanol by vortexing, subsequent centrifugation (4°C, 10 minutes, 12,000 xg) and discarding of the supernatant. RNA pellet was air-dried for 30 minutes and resuspended in 15 mikroliter RNAse-free water. Solubility of RNA was enhanced by incubation at 60°C for 10 minutes. Next-generation sequencing libraries were constructed from 1000 ng RNA with the Illumina TruSeq Stranded mRNA Kit, according to the manufacturer's instruction.
ENA-FIRST-PUBLIC
2020-02-12T04:04:55Z
ENA-LAST-UPDATE
2019-11-20T15:06:50Z
External Id
SAMEA6237230
INSDC center name
Friedrich-Alexander-University Erlangen-Nuernberg (FAU), Department of Internal Medicine 3 - Rheumatology and Immunology, Universitaetsklinikum Erlangen, Erlangen, Germany.
INSDC first public
2020-02-12T04:04:55Z
INSDC last update
2019-11-20T15:06:50Z
INSDC status
public
Submitter Id
E-MTAB-8536:08_V3_214707_Bari
broker name
ArrayExpress
cell type
mesenchymal stem cell of the bone marrow
common name
house mouse
developmental stage
adult
disease
normal
genotype
wild type genotype
growth condition
osteogenic induction
organism part
bone marrow
sample name
E-MTAB-8536:08_V3_214707_Bari
scientific_name
Mus musculus