GSM7116977: ASC52telo, TP53 KO, RB1 KO, PIK3CA OE, rep3; Homo sapiens; RNA-Seq
Total RNA was isolated from human sarcoma models and controls using the RNeasy Mini Kit (Qiagen) Library preparation was performed by the Functional Genomics Laboratory (FGL), a QB3-Berkeley Core Research Facility at UC Berkeley. Total RNA samples were checked on Bioanalyzer (Agilent) for quality. Only high- quality RNA samples (RIN > 8) were used. At the FGL, Oligo (dT)25 magnetic beads (Thermofisher) were used to enrich mRNA. The treated RNAs were rechecked on Bioanalyzer for their integrity. The library preparation for sequencing was done on Biomek FX (Beckman) with the KAPA hyper prep kit for RNA (now Roche). Truncated universal stub adapters were used for ligation, and indexed primers were used during PCR amplification to complete the adapters and enrich the libraries for adapter-ligated fragments. Samples were checked for quality on an AATI (now Agilent) Fragment Analyzer. Samples were then transferred to the Vincent J. Coates Genomics Sequencing Laboratory (GSL), where quantitative PCR was used to calculate sequence-able molarity with the Kapa Biosystems Illumina Quant qPCR Kits. Libraries were pooled evenly by molarity and sequenced on an Illumina NovaSeq6000 150PE S4. Raw sequencing data were converted into fastq format, sample-specific files using the Illumina bcl2fastq2 software.