title: 3D EV no DOX Exp1
organism: Homo sapiens
description: Protocols: DCIS.com cells containing the Doxycycline inducible pInducer21 empty vector (EV) or the pInducer21SOX11 were cultured in 2D and 3D for RNA isolation, as follows. For 2D cultures, 10^6 cells were plated in a p100 dish. After 24h they were cultivated in presence or absence of 1μM Doxycycline for 48h. For 3D cultures, 5000 cells grown either in absence or in the presence of 1μM doxycycline for 48h were used to form spheres in an ultralow attachment 96 well plates. 24h after plating (when the spheres are formed), the media was changed and half of the spheres formed in media without doxycycline were grown for 48h either in the absence of doxycycline (no DOX) or in presence of it (DOX 2days). Spheres formed in the presence of doxycycline were cultured for 48h more after changing media for a total of 5 days culture in doxycycline (DOX 5days). Total RNA from cells were extracted using miRNeasy Mini kit (217004 Qiagen, Manchester, UK). RNA samples were treated with Turbo DNA free Kit (AM1907 Thermo Fisher Scientific Waltham, MA, USA) to digest DNA and purified and concentrated with RNA clean and concentrator-5 (R1015 Zymo Research, Irvine, CA, USA). cDNA library preparation was carried out at Oxford Genomics Centre, The Wellcome Trust Centre for Human Genetics using PolyA+ RNA enrichment method for total RNA from cultured cells . mRNA fraction was selected from the total RNA before conversion to cDNA. Second strand cDNA synthesis incorporated dUTP. The cDNA was end-repaired, A-tailed and adapter-ligated. Prior to amplification, samples underwent uridine digestion. The prepared libraries were size selected, multiplexed and quality checked.