GSM2149110: RP_ZT8_1; ZT8, ribosome footprint, rep1; Mus musculus; RNA-Seq
Kidneys were homogenized in 3 volumes of lysis buffer (150 mM NaCl, 20 mM Tris-HCl pH7.4, 5 mM MgCl2, 5 mM DTT, 100 μg ml-1 cycloheximide, 1% Triton X-100, 0.5% Sodium deoxycholate, complete EDTA-free protease inhibitors (Roche) and 40 U ml-1 RNasin plus (Promega)) using a Teflon homogenizer. Lysates were incubated for 10 min on ice and cleared by centrifugation at 1000 x g, 4°C for 3 min in a tabletop centrifuge. Supernatants were flash-frozen and stored in liquid nitrogen. Lysates of two mice per timepoint were pooled. Total RNA-seq libraries were generated from the lysate pool. For ribosome profiling libraries, lysate pools (15 OD260) were digested with 650 U RNase I (Ambion) for 45 min at room temperature and passed over size exclusion columns for ribosome purification prior to RNA extraction and library preparation. Sequencing libraries for total RNA and ribosome protected RNA were generated using Ribo-Zero (Epicentre) and ARTseq ribosome profiling kits (Epicentre).
GEO Accession
GSM2149110