P-GSE39305-3 - P-GSE39305-3
nucleic acid library construction protocol
4x10^6 cells from each of the cells lines UMSCC25, 584-A2, and CCL30 were plated and transduced 24 hrs later with the SBI shRNA library (Mountain View, CA) and 8 μg/mL polybrene (Sigma-Aldrich) at a multiplicity of infection of ~0.2. After 72 hrs, transfected cells were selected with puromycin (1 μg/mL) for 5 days. Library expressing cells were divided into six groups of 5x106 cells per T-75 flask where three were treated with vehicle (DMSO) and the remaining three groups were treated with AZD12908010 (1.0 μM for UMSCC25, 0.125 μM for 584-A2 and 0.5 μM for CCL30). After 72 hrs, the cells were placed in fresh media without drug for an additional 72 hrs. Total RNA was collected from each replicate using a RNeasy Mini Kit (Qiagen, Germantown, MD) and reverse transcribed using vector specific primers and MMLV reverse transcriptase (Invitrogen, Carlsbad, CA). The cDNA was amplified using nested PCR to isolate the shRNA sequences and to add Illumina adapter sequences. The samples were then sequenced on an Illumina Genome Analyzer IIx (Illumina, San Diego, CA), and shRNA sequences were identified and quantified.