Computational protocol: Chromatin Dynamics and the RNA Exosome Function in Concert to Regulate Transcriptional Homeostasis

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Protocol publication

[…] Yeast were grown in yeast extract peptone (YEP) media with 2% glucose at 30°C. Total RNA was prepared, labeled, and converted into cDNA by random primed retrotranscription of total RNAs as previously described () before being hybridized to Affymetrix tiling microarrays. At least three biological replicates for each genotype were analyzed from three independent array hybridizations. Each array was normalized using W303 genomic DNA as reference (), and only transcripts scoring above a threshold background value were used for further processing, as previously published (). Expression level for each transcript was estimated by the midpoint of the shorth (shortest interval that covers half the values) of the normalized probe intensities lying within the transcript as previously described (), and differential gene expression analysis was performed using limma as detailed in . Microarray data can be viewed on the Steinmetz lab browser (http://steinmetzlab.embl.de/peterssonLabArray/). qRT-PCR was used to validate the results of the tiling array ().ChIP-seq samples were prepared () and analyzed either as in or by MACS2 () as described in . Two different biological samples were sequenced for each genotype. The 8WG18 antibody (Covance) was used for immunoprecipitations, as it is known to capture total RNA Pol II in genome-wide data (; ).The complete annotation used in this publication is listed in . This study focuses on five major groups of significantly changed (padj = FDR < 0.1 and log2 fold change [LFC] > ± 0.59) transcripts defined below:Group A ORFs are (1) significantly upregulated in rrp6Δ compared to WT and (2) reduced by > −0.59 LFC in rtt109Δ rrp6Δ compared to rrp6Δ. Refer to , , and and .Group B ORFs are (1) significantly downregulated in rrp6Δ compared to WT and (2) increased by > 0.59 LFC in rtt109Δ rrp6Δ compared to rrp6Δ. Refer to , , and and . This group includes ORFs subject to transcriptional interference by adjacent CUTs.Group C CUTs are (1) significantly upregulated in rrp6Δ compared to WT and (2) reduced by > −0.59 LFC in rtt109Δ rrp6Δ compared to rrp6Δ. Refer to , , and and .Group D CUTs are (1) significantly upregulated in rrp6Δ compared to WT and (2) reduced by > −0.59 LFC in rtt109Δ rrp6Δ as well as swr1Δ rrp6Δ compared to rrp6Δ. Refer to , , and and . Up_ncRNAs are (1) significantly upregulated in swr1Δ rrp6Δ compared to rrp6Δ and include SRTs (n = 45), novel (n = 100), SUTs (n = 50), and CUTs (n = 29). Refer to , , and and . Unchanged_ncRNAs (n = 485) are SRTs that do not change significantly in swr1Δ rrp6Δ compared to rrp6Δ. Refer to and . […]

Pipeline specifications

Software tools limma, MACS
Application ChIP-seq analysis
Organisms Mus musculus, Saccharomyces cerevisiae
Diseases Chromosome Aberrations