Illumina HiSeq 2500 sequencing; Analysis of ribosomal occupancy with ribosome profiling.
Analysis of ribosomal occupancy with ribosome profiling.
Cells were not treated with siRNA (untreated), treated with anti-STAU1 siRNA (knockdown), or treated with anti-STAU1 siRNA and the STAU1 level was rescued by the expression of siRNA-resistant STAU1 (rescue). Cells were subjected to Cycloheximide treatment before collection. Ribosome profiling protocol was followed to isolate protected RNA from the Cycloheximide teated cells and amplify cDNA library as described in the manuscript. Specific barcodes were used for each RT reaction as described in the manuscript. Random barcodes were also incoperated into cDNAs to distinguish between PCR duplication of cDNA products. The final products were obtained by PCR with Illumina paired-end sequencing primers: 5-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3; 5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3
Experimental Factor: treatment
rescue
LIBRARY_STRAND
first strand