title: GSM5129836: MV90_D25_Ctrl_3; Mus musculus; RNA-Seq
library_construction_protocol: Samples were taken at Day 20 (Ctrl only), 22, 25 and 30 of differentiation. At each timepoint per experimental condition 6 individual organoids were sampled. Organoids were lysed in 100µl TRIzol by mortar-pestle homogenization, RNA was isolated using miRNeasy Kit (Qiagen) and Maxtract High Density tubes (Qiagen), residual DNA was removed using Ambion TURBO DNA-free Kit (Thermo Fisher) and RNA cleaned up by ethanol-precipitation. The RNA integrity for each sample was controlled with the RNA 6000 Nano Assay and the Agilent 2100 Bioanalyzer (Agilent Technologies). 100ng total RNA was used for rRNA depletion. Ribosomal RNAs were removed from total RNA. A strand-specific library using the Ribo-Zero Gold H/M/R Magnetic Kit (Illumina, San Diego, CA, USA). A strand-specific library was prepared using the ScripSeqv2 Kit (Illumina), was checked by Agilent 2100 Bioanalyzer system with a High Sensitivity DNA Kit (Agilent). Library concentration was determined by Qubit 2.0 instrument using the Quant-iT dsDNA High Sensitivity kit (Thermo Fisher Scientific).  size selected in a range of 200-600 bp using preparative agarose gel in combination with MinElute Gel Extraction Kit (Qiagen).