GSM7634916: PDM50_PF_treated_S74_L001; Homo sapiens; RNA-Seq
To collect CRC PDM organoids for analysis of gene expression in response to PF-06409577, dissociated single cells were seeded in Matrigel in 24-well tissue culture plates. After 3 days in culture, organoids were administered complete medium or complete medium treated with 10 µM PF-06409577. Prior to treatment with PF-06409577 and after 2 days of treatment, whole organoids were collected in Dulbecco's phosphate-buffered saline with 0.5 mM ethylenediaminetetraacetic acid (PBS-EDTA). Organoids were subsequently incubated in cell recovery solution, pelleted, and stored at -80ºC. Total RNA was extracted using the Quick-RNA Microprep Kit and concentration was measured using Nanodrop OneC Spectrophotometer. For qPCR, first-strand cDNA synthesis was performed using qScript cDNA SuperMix. RNA sequencing libraries were generated using the Illumina TruSeq Stranded Total RNA Library Prep Gold with TruSeq Unique Dual Indexes (Illumina, San Diego, CA). Samples were processed following manufacturer's instructions, except modifying RNA shear time to five minutes. Resulting libraries were multiplexed and sequenced with 100 basepair (bp) Paired End (PE100) to a depth of approximately 25-40 million reads per sample on an Illumina NovaSeq 6000. Samples were demuxltiplexed using bcl2fastq v2.20 Conversion Software (Illumina, San Diego, CA).