title: Illumina HiSeq 2500 sequencing; Using ribosome profiling to investigate the effect of p53 on translation in cancer cells
design: Using ribosome profiling to investigate the effect of p53 on translation in cancer cells
library_construction_protocol: Prior to collection cells were treated with 1 microgram/ml doxycycline (for Tetracycline-controlled transcriptional activation) and 1 microM doxorubicin (to activate p53) for 19 hours, and with 100 microgram/ml cycloheximide for the last 10 minutes. The cells were lysed on plate, scraped, centrifuged, and the supernatant was used for RNA extraction. Three cell lines were established using H1299 cells with a Tet-on expression system with an empty response vector (control), with a response vector expressing p53 WT (WT), and with a response vector expressing mutated (R175H) p53 (mutant). Cells were lysed and RNA was purified by TRI Reagent (Sigma) and PureLink RNA Micro Kit (Invitrogen). Samples were treated with Ribo-Zero Gold rRNA removal Kit (Illumina) for rRNA depletion. Libraries were generated using the TruSeq Ribo Profile (Mammalian) Kit from Illumina.   Heat fragmented total RNA and size-selected RPF RNA samples were end-repaired, 3' adapter ligated and reverse transcribed. Resulting cDNA samples were PAGE-purified, circularised, and PCR-amplified with Index primers.