title: Sample 1
organism: Homo sapiens
description: Protocols: Briefly, U87MG-GFP cells were implanted sub cutaneously into dorsal flanks of male NOD/SCID mice (6-8 weeks) and allowed to grow for three weeks. Hoechst 33342 (vascular perfusion dye) was injected as onto the tail vein and the tumor resected post 20min incubation. Tumors were isolated, immediately dissociated into single cells. Single cell suspensions were sorted using BD FACSAria™ III. The gating strategy was used to isolate only GFP+ cancer cells (to exclude all stromal elements) and these cells were further fractionated into three fractions - Hh (Hoechst High), Hm (Hoechst medium) and Hl (Hoechst low) based on their dye uptake. Hh - are the cells closest to blood vasculature and Hl are the farthest. 50000 sorted cells were centrifuged and resuspended in Trizol. Total RNA extraction was performed according to manufacturer's protocol (qiagen, RNeasy Mini Kit #74104). Library construction was performed according to CelSeq2 protocol (Hashimshony T et al., Genome Biology, 2016).