title: GSM8000276: Mutnat untreated astrocytes, RNA-seq, batch A; Mus musculus; RNA-Seq
library_construction_protocol: After thorough removal of PBS 230ul of lysis buffer were added to each 15 cm plate. The lysis buffer, composed of 25mM K-HEPES pH=7.2, 10mM MgCl2, 200mM KCl and freshly added 2% DDM, 1mM DTT, 1X complete EDTA-free protease inhibitor (Roche), 100 μg/ml cycloheximide, and 25U/ml Turbo DNase-I (Thermo Scientific, AM2238), was added directly onto the cells for 5 min incubation on ice. Cell lysates were scraped and collected, then incubated on ice for additional 10 min. A portion of each sample was treated with RNase-I (Thermo Scientific, AM2295) to generate ribosome footprints, while an undigested portion was used to isolate total RNA. Following RNase treatment, the footprint samples were layered on 1M sucrose cushion, following ultracentrifugation at 78,000 rpm for 2 h at 4°C in a Beckman Coulter TLA120.2 rotor. RNA extracted from sucrose cushion pellets were subjected to size selection, by separation of the footprint samples on a 15% PolyAcrylamide TBE-urea gel followed by purification of 26-34 nt fragments from the gel. libraries for RNA-seq were prepared using TruSeq Stranded Total RNA Library Prep Gold kit (Illumina #20020598) following manufacturer's protocols. libraries for Ribo-seq were generated with NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, #E7300L), then size selected by gel electrophoresis on 8% PolyAcrylamide Gel, with ~155 bp sized product purified from the gel.