GSM1184594: 1.5h_MM1S_20nM_bortezomib_ribosome_footprint; Homo sapiens; RNA-Seq
Ribosome footprints were isolated from 80S ribosomes by ultracentrifugation followed by gel-based size selection, as previously described (Ingolia et al, Cell 2011). Poly(A) mRNA was isolated using poly-d(T) coated magnetic beads, then size-selected by PAGE after alkaline fragmentation. Libraries were constructed as previously described (Ingolia et al, 2011). Briefly, size-selected ribosome footprint and poly(A) mRNA fragments were ligated to Linker-1 oligonucleotide (IDT) by T4 RNA ligase following PNK dephosphorylation. Ligated RNA was purified after gel-based separation.  Reverse transcription to cDNA was carried out using SuperScript III enzyme. cDNA fragments were purified after gel-based separation. Ribosomal RNA subtraction was performed on ribosome footprint samples using biotinylated oligonucleotides (listed in Stern-Ginossar et al, Science 2012) and streptavidin-coated magnetic beads.  Samples were circularized using Circ Ligase (Epicentre) and PCR amplified using primers containing Illumina barcodes for final libraries. Up to four samples were multiplexed in a single lane for sequencing.
GEO Accession
GSM1184594