title: GSM3098498: Ribosome_footprint_H2O2_Rep2_0hr; Homo sapiens; OTHER
library_construction_protocol: For RNA seq and ribosome foot print experiment, confluent plates of HeLa cells were first aspirated of their growth media and washed with fresh media supplemented with 0.1 mg/ml cycloheximide. The media was then removed and the cells were washed with 10 ml chilled PBS containing 0.1 mg/ml cycloheximide. Following removal of the PBS, 800 ul of Lysis Buffer was added and the cells were extensively scraped off plate and transferred to a pre-chilled eppendorf tube, recovering ~1 ml of lysate per sample. To insure complete lysis, we passed the lysate through a 25 gauge needle and further incubated on ice for ~10 min. The lysate was then clarified by centrifugation for 10 min at 20,000, 4°C and ∼1 ml supernatant was recovered. For each treatment (tunicamycin and hydrogen peroxide), we aliquoted 100 ul of the cell lysate for total RNA extraction and 200ul for ribosome footprinting. For protein foot print, we scraped crosslinked cells off the plates with a rubber policeman, collected by centrifugation, washed with ice-cold PBS . We resuspended cell pellets in lysis/binding buffer (100 mM Tris-HCl pH 7.5 at 25 °C, 500 mM LiCl, 10 mM EDTA pH 8.0 at 25 °C, 1% LiDS, 5 mM DTT, Complete Mini EDTA-free protease inhibitor (Roche)), incubated them at room temperature for 15 minutes for lysis and passed cells through a 21 gauge needle 10 times for shearing of genomic DNA. Lysates were processed to recover RNA-protein complex as described previously (Munschauer, 2015). RNA libraries for RNA seq, Ribosome foot print and protein foot print were prepared according to the TruSeq Ribo Profile (Mammalian) kit protocol.
attributes.tag: GEO Accession
attributes.value: GSM3098498