title: GSM8839409: Leucine and Isoleucine deprivation, rep1; Mus musculus; OTHER
library_construction_protocol: Cells were washed with cold PBS containing cycloheximide and lysed in polysome lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1% Triton X-100, 5 mM DTT, 100 µg/ml CHX, DNase, RNase inhibitor, protease/phosphatase inhibitors). Lysates were cleared by centrifugation. Ribosome-protected fragments were generated by RNase I digestion. Digested samples were purified using Microspin S-400 HR columns (Cytiva) equilibrated with polysome buffer, RNA extracted by phenol-chloroform extraction and ethanol precipitation,  and size-selected (26–34 nt) on 15% TBE-Urea gels. rRNA-depletion was performed with biotinylated oligos and Streptavidin Dynabeads. Samples were purified using a RNA Clean & Concentrator kit (Zymo). End repair was performed by incubating RNA with T4 PNK (NEB) at 37°C for 1 hour, followed by ATP addition and another hour of incubation. Clean-up was performed using the  RNA Clean & Concentrator kit (Zymo). Libraries were prepared using the NEXTflex Small RNA-Seq Kit v3 (Revvity).
identifiers.namespace: GEO
identifiers.id: GSM8839409_r1