GSM5462544: BP8-Primary control (TNFR1-/+) CMFs-MS2; Mus musculus; RNA-Seq
Control (TNFR1+/-) and TNFR1-/- primary CMFs (passage 6 - 8) were seeded at a density of 1x103 cells/50 µL of Matrigel into 24-well plates and were grown to 70% confluency. At D 5, CMFs were extracted from Matrigel after brief incubation at 4⁰C via centrifugation at 1200rpm for 5 min. Total RNA from primary CMFs was extracted with using the Ambion® PureLink® RNA Isolation Kit (Thermofisher Scientific, no. 12183025) according to the manufacturer's protocol. mRNA is purified from approximately 500ng of total RNA with oligo-dT beads and sheared by incubation at 94C.  Following first-strand synthesis with random primers, second strand synthesis is performed with dUTP for generating strand-specific sequencing libraries.  The cDNA library is then end-repaired, and A-tailed, adapters are ligated and second-strand digestion is performed by Uricil-DNA-Glycosylase. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul are used for sequencing.