GSM8179578: D3_MSCV-backbone_DN1; Homo sapiens; RNA-Seq
Cells were counted and lysed with 800 µl to 1 ml TRIzol (15596026, ambion) reagent, incubated for 10 minutes at RT and stored at -80 °C. Chloroform (C2432-26ML, Honeywell) was added at 160 µl or 200 µl respectively, to defrosted samples followed by vigorous shake for 15 seconds and 3 minutes at RT incubation. A centrifugation step at 11,000 rpm for 15 minutes at 4 °C was followed by collection of the aqueous phase. 400 µl isopropanol and 10 µl glycogen (AM9510, Invitrogen) were added and the mixture was incubated for 10 minutes at RT. Samples were centrifuged at 11,000 rpm for 10 minutes at 4 °C and the pellet was washed three times with 75% ethanol followed by a centrifugation step at 9,000 rpm for 5 minutes at 4 °C. RNA pellets were air dried for 10 minutes and dissolved in 30 µl RNase free water. RNA was incubated at 55 °C for 10 minutes, treated with DNases for genomic DNA removal (AM1906, Invitrogen) and stored at -20 °C. Sequencing libraries and sequencing were carried out by Novogene.