Additional replicate mRNA seq libraries were prepared with yeast strains grown under normal conditions with out the addition of 4sU. Total RNAs were extracted with acid phenol chloro form, pH 4. 5 with isoamyl alcohol, 25 24 1. Replicate, strand specific total mRNA seq libraries were http://www.selleckchem.com/products/Belinostat.html prepared in parallel using the two linker ligation protocol as described. For preparation of ribo mRNA seq libraries, extract preparation, and ribosome depletion using sucrose den sity gradients were carried out as described in the gPAR CLIP procedure. PolyA mRNAs were enriched using oligo 25 beads and converted into sequencing libraries as described. Data processing of Illumina HiSeq sequencing reads gPAR CLIP, PAR CLIP, and mRNA seq reads were pro cessed to remove linkers, sorted into libraries based on six nucleotide barcodes, and removed if they were low quality.

First, reads with a perfect match to a barcode were suc Inhibitors,Modulators,Libraries cessfully sorted, followed by reads with one mismatch to a barcode. If both barcodes were perfectly Inhibitors,Modulators,Libraries matched in a read or both barcodes were found with one mismatch, the 3 most barcode was chosen. 99. 97% of reads were successfully sorted into libraries under these rules. Next, reads were removed if they met any of the following cri teria 18 nucleotides, only homopolymer As, missing 3 adapter, 5 3 adapter ligation products, 5 5 adapter ligation products, low quality. 97. 0% of gPAR CLIP, Inhibitors,Modulators,Libraries 80. 0% of PAR CLIP, and 99. 7% of mRNA seq reads passed these filters. High quality reads were mapped to the S. cerevi siae genome version S288C with Bowtie using the following parameters v 3, k 275, best, and strata.

Mapped reads were annotated using custom scripts to known genomic elements in the S288C gen ome including external UTR annotations. Additional Inhibitors,Modulators,Libraries file 1 provides read counts at each processing step. Assessing data reproducibility To determine mRNA seq and gPAR CLIP replicate library reproducibility, we calculated replicate correla tion using normalized read counts of each gene. Pearson correlation coefficients for mRNA seq libraries ranged from 0. 984 to 0. 994, while coefficients for gPAR CLIP libraries ranged from 0. 967 to 0. 971. Due to high reproducibility, Inhibitors,Modulators,Libraries subsequent measures of read coverage represent averages of two biological replicate libraries. To determine if the addition of 4sU to growth media substantially alters transcription, biological replicate mRNA seq libraries were generated from wild type yeast grown under normal conditions without the addition of 4sU. These replicate libraries had a Pearson correlation coefficient of 0. 988, indicating high reproducibility, and a Pearson correlation selleck chem inhibitor coefficient of 0. 982 when com pared to wild type yeast grown in the presence of 4sU.