GSM7987640: GSC-20, M2 Macrophage CM, Day 28, invasive fraction_rep2; Homo sapiens; RNA-Seq
All samples were treated with 10k U/ml Hyaluronidase from bovine testes, Type IV-S (Sigma-Aldrich, H3884) for 15 min with agitation until the hydrogel was fully degraded. Afterwards, RNA was extracted using TRIzol Reagent (Invitrogen, 15596018) according to manufacturer's recommendations. In brief, 1M cells were mixed with 100 µl TRIzol Reagent and the solution was vortexed for 30 seconds. 20 µL chloroform (Sigma, c2432) was added to the mixture and the solution was placed on ice for 15 minutes. Sample were centrifuged at 14000 rpm for 10 minutes and the clear RNA layer was carefully transferred to a new Eppendorf tube. Each RNA sample was mixed with a 1:1 volume of 2-Propanol (Sigma-Aldrich, 190764-4L) and 0.5 µL Glycogen, RNA grade (Thermo Scientific, R0551). After an overnight incubation at -20˚C, samples were centrifuged at 14000 rpm for 10 minutes at 4˚C and the RNA pellet was rinsed twice by aspirating the supernatant, adding 400 µL 75% Ethanol and centrifuging at 14000 rpm for 1 minute. Following the second rinse, the supernatant was carefully and completely removed, and RNA pellets were incubated at room temperature until visibly dry. RNA was dissolved in 30 µL RNase-free DNase-free distilled H2O and RNA concentration and purity was measured with Nanodrop. Isolated RNA was sent to Novogene Corporation Inc. (Sacramento, CA) for library construction, quality control and sequencing. In brief, messenger RNA was purified 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.