GSM6647341: MDMs control 6 h biol rep 1; Homo sapiens; RNA-Seq
3-5*10^5 MDMs were washed once with PBS. Trifast reagent (Peqlab) was added to the cells together with an amount of 10% of Drosophila Schneider S2 cells relative to MDMs; total TRIfast volume was 500 µl. The pellet was fully resuspended and incubated for 5 min at RT in an 1.5 ml Eppendorf tube. 100 µl of chloroform were added, and the tube was shaken by hand for 15 s. After 3 min of incubation at RT, the tube was centrifuged at 12.000g at 4 degrees centigrade. The upper phase was transferred to a fresh tube, and 2 µl of Glycoblue (Thermo Fisher Scientific) were added followed by 250 µl of isopropanol. After mixing by shaking, the sample was incubated for 10 min at RT followed by centrifugation at 12.000g at 4 degrees centigrade. The supernatant was discarded, and the pellet was washed with 500 µl of 70% (v/v) ethanol. The supernatant was discarded, the pellet was air-dried and subsequently dissolved in 40 µl of water supplemented with 20 U of SUPERase-In RNase inhibitor (Thermo Fisher Scientific). A synthetic ERCC RNA spike-in mix (Thermo Fisher Scientific) was added to the sample according to the manufactur The QuantSeq FWD Kit (Lexogen) was used according to the manufacturer's instructions.