GSM3378855: 32873 Non-Diff mRNA; Homo sapiens; OTHER
Cells were washed with ice cold PBS with CHX at 100ug mL-1. Plates were immediately flash frozen in liquid nitrogen, moved to dry ice, and lysed (in the presence of CHX) to prevent ribosome loading and runoff. Additional lysates were processed in parallel for poly(A) mRNA purification  and  mRNA-sequencing  library  preparation.  Polysomes  were  isolated  from  the ribosome footprinting lysates on a 1M sucrose cushionwith high speed centrifugation using a 70.1Ti rotor (Beckman) at 55,000 r.p.m. for 4 hrs at 4°C. rRNA was eliminated prior to linker ligation using Ribo-Zero Gold rRNA Removal Kit (Illumina). Ribosome profiling libraries were prepared as in Ingolia et al., 2010 and Ingolia et al., 2012. Ribosome Profiling libraries were barcoded and multiplexed with2-4 libraries per lane, and sequenced on a HiSeq 2000 (Illumina) using 50 cycles of single end reads. mRNA libraries were multiplexed on a single lane. All sequencing was conducted at the University of Michigan DNA Sequencing Core.
GEO Accession
GSM3378855