GSM1655066: Ribosome Profiling puromycin replicate 2; Mus musculus; OTHER
After drug treatment, murine ES cells (CGR8) were washed twice with ice cold PBS, 100 µg/ml Cycloheximide, scraped, pelleted and suspended in 200 µl ice cold 50 mM Tris/Cl pH 7.5, 100 mM KCl, 6 mM MgCl2, 5 mM DTT, 100 µg/ml Cycloheximide, 0.25 M Sucrose. Cells were lysed by adding 200 µl of the same buffer with 1% NP40 and 1% Deoxycholate. Nuclei and the bulk of mitochondria were removed by two 7 min 10,000 g centrifugations at 4 °C. 100 µl of the lysate was used directly for preparation of total RNA. To the rest, 40 U RNAseI (Fermentas), 4U DNaseI (Promega, WI) were added and incubated for 1 hr at 30 °C. Monosomes were pelleted by a 2 hr centrifugation at 140,000 g and RNA isolated using a Qiagen small RNA isolation kit. Ribosomal RNA was depleted with biotinylated antisense cRNA covering the murine 28S and 18S rRNA sequences and biotinylated antisense oligonucleotides complementary to the 5.8S and 5S rRNA.  For non RNAse treated samples, RNA was fragmented in Ambion RNAseIII buffer for 8 min at 95°C. RNA fragments from the heat fragmentation or RNAse I digestion were kinased with polynucleotide kinase. Libraries were prepared with the New England Biolabs NEBnext small RNA library preparation kit for Solid. stranded RNA-seq , adapter ligation to fragmented RNA ( New England Biolabs NEBnext Small RNA Library prep kit) SOLiD5500 Wildfire, Life Technology. Sequencing of 50b. with 10b. barcoding sequences.
GEO Accession
GSM1655066