title: Effect of PAX3-WWTR1 knockdown in rhabdomyosarcoma derived tumoroids
design: Effect of PAX3-WWTR1 knockdown in rhabdomyosarcoma derived tumoroids
library_construction_protocol: For RNA isolations 2 wells were washed briefly with icecold PBS and cells were lysed directly in TRIzol (ThermoFisher Scientific 15596026 ). For Immunoblotting 2 wells were washed with PBS and cells were lysed directly in 2xLaemmli sample buffer with β-mercaptoethanol.  RMS000EEC organoids were cultured as described in Meister et al. (55) Briefly early passage cryovials were restarted at high density (50,000 cells/cm2) in BM1* medium supplemented with 0.3% BME. Cells were growing at 37o C and 5% CO2 as attached cells with a doublingtime of 144 hrs, and passaged to start from 25,000 cells/cm2. siRNA transfections were performed by harvesting RMS000EEC cells and replating in 24 well plates at 200,000 cells per well in 500ul BM1* with 0.3% BME. After 24 hours transfections were done by adding to each well a 50ul mixture of Lipofectamine RNAiMAX (ThermoFisher Scientific 13778100) and DsiRNA in Optimem (ThermoFisher Scientific 31985062), such that the endconcentration in the well is 3ul LF/ml, 30nM DsiRNA . Cells were washed after 24 hours and harvested after 48 hours.  NEBNext® UltraTM RNA Library Prep Kit (New England Biolabs) was used, according to manufacturer's instructions, to prepare the sequencing libraries. Strand specific mRNA libraries were prepared by Novogene (UK). RNA samples were sequenced using a paired-end approach