title: 03_V1_214702_Tofa
organism: Mus musculus
description: 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.