title: GSM8376546: mmu_ptch_EZH2i_1; Mus musculus; RNA-Seq
library_construction_protocol: Primary cells were freshly isolated from tumor-bearing mice at 6 to 8 weeks of age. Briefly, tumor tissues were isolated from mouse brains and digested in a papain solution to obtain a single cell suspension and then centrifuged through a 35% and 65% Percoll gradient. Cells from the 35% to 65% interface were suspended in Dulbecco's PBS (DPBS) plus 0.5% BSA. Cells were then suspended in NB-B22 (Neurobasal medium with B22 supplement. Cell suspension was plated on poly-D-lysine (PDL)-coated coverslips (BD Biosciences). Total RNA was extracted from tumor cells treated with T3 or PBS. Strand-specific mRNA-seq libraries for the Illumina platform were generated and sequenced following the manufacturer's protocol. High-quality total RNA was used as input for the so-called dUTP library preparation method. Briefly, the mRNA fraction was purified from total RNA by polyA capture, fragmented and subjected to first-strand cDNA synthesis with random hexamers in the presence of Actinomycin D. The second-strand synthesis was performed incorporating dUTP instead of dTTP. Barcoded DNA adapters were ligated to both ends of the double-stranded cDNA and subjected to PCR amplification. The resultant library was checked on a Bioanalyzer (Agilent) and quantified. The libraries were multiplexed, clustered, and sequenced on an Illumina NovaSeq X Plus. The sequencing run was analyzed with the Illumina BCL-Convert, with demultiplexing based on sample- specific barcodes.