GSM9245560: Neutrophil HG RNA-seq replicate 4; Homo sapiens; RNA-Seq
Neutrophil mRNA were purified from 5x107 neutrophils either freshly purified or cultured for 18h in anoxia in RPMI 1640 medium supplemented with 10 mM Hepes and 3 mM glucose. Cells were washed once with PBS and centrifuged for 5 minutes at 300 x g. Pelleted cells were lysed in 350 µL RLT buffer (Qiagen). Lysates were resuspended with a needle to increase the lysis efficiency. TRIzol (3 volumes) was added to samples followed by vortex and short spin. 1/5 of the volume of chloroform was added and samples were vortexed and incubated for 3 minutes at room temperature prior to transferring into PLG heavy tubes. Samples were centrifuged for 15 min at 12.000 x g (4°C) and RNA-containing aqueous solution was transferred into a new tube. RNA was purified using the RNeasy Mini Kit (Qiagen) and quantified with a nanodrop, and the quality was assessed with Tapestation. RNA samples were analysed and sequenced by the GENOM'IC platform (Institut Cochin, Paris, France)