Cells were lysed on plate in 15 mM Hepes pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton-X100. Clarified lysates were split in two to make libraries for ribosome profiling and RNA-seq. For ribosome profiling lysates were RNase I treated and centrifuged through a sucrose cushion. RNA was extracted from the resulting ribosome pellet by proteinase K treatment and acid phenol extraction. rRNA was removed with the Ribo-Zero Gold kit. 28-34 nt footprints were then isolated on polyacrylamide gel, dephosphorylated followed by 5' adapter ligation (in case of the double knock out samples, this adapter contained a unique molecular identifier and an additional barcode) and reverse transcription. The resulting cDNA was circularized and PCR amplified, incorporating Illumina sequences. For RNA-seq on WT cells RNA was extracted from the lysate by proteinase K digestion followed by phenol acid extraction and fragmented by alkaline hydrolysis. 40-80 nt fragments were purified and libraries were further prepared similar to the RPF libraries. For sgLARP1 and sgLARP1/1B cells, RNA-seq libraries were prepared using the NEB Next Ultra II Directional RNA Library Prep kit along with the NEBNext rRNA Depletion kit.
GEO Accession
GSM3889091