GSM1887643: ribosome profiling; Homo sapiens; miRNA-Seq
For cell lysis, the cell medium was aspirated and cells washed with ice-cold PBS containing 100 µg/ml cycloheximide. No cycloheximide was added to the culture medium before. After thorough removal of the PBS, the plates were immersed in liquid nitrogen and placed on dried ice. For cell lysis, 400 µl mammalian polysome buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, with 1 mM DTT and 100 µg/ml cycloheximide added freshly) was supplied with 1% (v/v) Triton X-100 and 25 U/ml Turbo DNase (Life Technologies, AM2238) and dripped on the plate which is subsequently placed tilted on wet ice. The cells were scraped off to the lower portion of the dish so that they thawed in lysis buffer. After dispersal of the cells by pipetting, the lysate was triturated ten times through a 26G needle, cleared by centrifugation at 20000g for 5 minutes, flash-frozen in liquid nitrogen and stored at -80 °C until further usage. For isolation of ribosome-protected RNA fragments, 120 µl of the lysate were digested with 3 µl RNase I (Life Technologies, AM2294) for 45 min. at room temperature with rotation. The digestion was stopped by addition of 4 µl Superase-In (Life Technologies, AM2694). Meanwhile, MicroSpin S-400 HR columns (GE Healthcare, 27-5140-01) were equilibrated with 3 ml mammalian polysome buffer by gravity flow and emptied by centrifugation at 600g for four minutes. Of the digested lysate, 100 µl were then immediately loaded on the column and eluted by centrifugation at 600g for two minutes. RNA was extracted from the flow-through, approximately 125 µl, using Trizol LS (Life Technologies, 10296-010). Ribosomal RNA fragments were then removed using the RiboZero Kit (Illumina, MRZH11124) and separated on a 17% denaturing Urea-PAGE gel (National Diagnostics, EC-829). The size range from 27nt to 30nt as defined by loading 20 pmol each Marker-27nt and Marker-30nt was cut out and the RNA fragments subjected to smallRNA library generation using 3'-Adapter NN-RA3, 5' adapter OR5-NN, RT primer RTP and PCR primers RP1 (forward primer) and RPI6-7 (reverse primer, containing barcodes). Libraries were sequenced on a HiSeq 2000 device (Illumina). After initial quality control, we obtained ~29 Mio raw reads by pooling the RPI6 and RPI7 samples.
GEO Accession
GSM1887643