GSM3336490: Disome9_RPF; Saccharomyces cerevisiae; OTHER
Ribosome protected fragments were dephosphorylated at the 3' end and cDNA was synthesized using the SMARTer smRNA-Seq kit (Clontech). cDNAs were depleted of rRNA-derived fragments by selective hybridization to biotinylated oligonucleotides. cDNA was then PCR amplified. Cell lysis was performed as described in detail previously (Li et al., Cell 2014), with the following modifications. 500 mL of cell culture was rapidly filtered at 30C by passing through a nitrocellulose filter. Cell pellets was were rapidly collected using a pre-warmed metal spatula, flash frozen in liquid nitrogen, and combined with frozen droplets of lysis buffer. Cells and lysis buffer were pulverized in 25 mL canisters (Retsch) pre-chilled in liquid nitrogen using Qiagen TissueLyser II. Pulverized lysate was thawed on ice and clarified by centrifugation at 4C. Lysates were treated with RNase I and ribosome protected fragments were isolated by hot phenol extraction. Ribosome protected fragments were size selected by gel extraction.
GEO Accession
GSM3336490