title: Illumina HiSeq 2500 paired end sequencing; Packaging of DNA:RNA hybrids in extracellular vesicles and their horizontal transfer in cancer
design: Packaging of DNA:RNA hybrids in extracellular vesicles and their horizontal transfer in cancer
library_construction_protocol: ZR751 and BT474 cell lines were purchased from the American Type Culture Collection (ATCC). All cells were mycoplasma free and maintained in MEM and RPMI (ATCC and MSKCC Media Core) supplemented with 5% fetal bovine serum (Media Core), 2mM glutamine, 100units ml-1 penicillin, and 0.1mg ml-1 streptomycin (Media Core). To precipitate and isolate the RNA fraction from the Hybrid and Non-Hybrid fractions, 500μl of trizol were added to both to the Non-Hybrid and the Hybrid solution and mixed by inverting the tubes and centrifuged 30 seconds at 12,000g. 200μl of chloroform were added to the samples, mixed by inversion and incubated for 2-3' min at RT. After a centrifugation at 12,000xg for 15' min at 4°C, the upper phase was transferred to a new tube containing 400μl of isopropanol and 3μl of glycogen. Samples were incubated overnight at -20°C. followed by an incubation of 10' min at room temperature and centrifuged at 12,000xg for 20' min at 4°C. The supernatant was discarded and the pellet was washed in 750μl of 75% EtOH. RNA was precipitated by centrifugation at 8,000xg for 20' min at 4°C. Pellet was then resuspended in 10μl of DEPC H2O and incubated for 5' min at 65°C. RNA concentration was measured by loading 1μl of DNA on a Nanodrop 1000 Spectrophotometer. Samples were stored at -80°C. RNA was isolated from ZR751 (acceptors/responders) and BT474 (non acceptors/non responders) following education with EV-Hybridlow and EV-Hybridhigh (each sample was run in duplicate). 750ng of total RNA was depleted for ribosomal RNA according to standard procedure and used for Illumina Truseq RNA library preparation and sequencing.