NextSeq 500 sequencing; RNA-seq of co-culture experiment between human HUVEC endothelial cells and mouse glioma GL126 cells
RNA-seq of co-culture experiment between human HUVEC endothelial cells and mouse glioma GL126 cells
HUVEC-RFP (HR) cells were plated overnight in EGM-2 medium in T75 flask along with Gl261-GFP (GG) at a seeding density ranging from 100,000 to 500,000 cells such that they reach a ratio of 3:1 or 1:3 (HR: GG) post 72 hours. The cells were trypsinized and suspended in fresh EGM-2 medium. A small fraction of it was subjected to FACS analysis to establish the exact ratio between HR and GG. For appropriate control (Sep), HR and GG were grown separately in EGM-2 medium and were mixed in the identical ratio as that of co-cultured cells. The samples were quickly lysed and RNA was extracted for transcriptomics.  RNA from the cells was extracted using Trizol reagent (Sigma). The cDNA synthesis was performed using RNeasy mini kit (Qiagen).  ). Libraries were prepared and sequenced using Illumina directional RNA sequencing protocol (Hi-Seq).