GSM9140069: Cancer tissue, coculture with cardiac tissue, bio rep 1; Homo sapiens; RNA-Seq
RNA extraction and isolation of the tissues cultured in AngioPlate was performed using the PicoPure™ RNA Isolation Kit (Applied Biosystems, KIT0204) according to manufacturer's protocol. Briefly, the cancer tissue in the middle well of the AngioPlate was removed and placed in a RNase-free microtube, followed by adding 100 µL of extraction buffer, incubating at 42ºC for 30 minutes, and centrifuging at 3000g for 2 minutes to collect the supernatant containing RNA. The RNA extract from each sample was mixed with equal volume of 70% ethanol by pipetting several times until a uniform mixture was formed. The mixture was then run through a preconditioned column to have RNA bound to the column, followed by 3-4  washes according to the manufacturer's instructions. Lastly, 12 µL of elution buffer was used to elute the RNA from the column. Quantification of isolated RNA was performed using a NanoDrop spectrophotometer. Isolated RNA was stored at -80ºC before sequencing. Non strand specific library Messenger RNAwaspurified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Strand specific library Messenger RNAwaspurified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. Then the second strand cDNA was synthesized using dUTP, instead of dTTP. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.