Computational protocol: Cell cycle population effects in perturbation studies

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Protocol publication

[…] Deletion strain DNA microarray data (Fig ) are from Kemmeren et al (), and these data are available in ArrayExpress (accession numbers E-MTAB-1383, E-MTAB-1384), GEO (accession numbers GSE42526, GSE42527) as well as in other formats described in the original study. Deletion strain data were generated as four replicates: two biological replicates harvested in early mid-log phase in synthetic complete medium with 2% glucose and each profiled in technical replicate. These were averaged and statistically compared to the average of over 400 wild-type expression profiles generated in parallel (Kemmeren et al, ). Expression data of strains grown in deletion strain conditioned medium (Fig ) have been deposited in ArrayExpress (E-MTAB-2218) and GEO (GSE54539), as has the heat shock profile (Fig , E-MTAB-2219, GSE54528), the heat shock time course (Fig , E-MTAB-2219, GSE54528), the low phosphate response (Fig , E-MTAB-2217, GSE54527), and the cold shock time course (, E-MTAB-2219, GSE54528). Growth of strains for the experiments specific to this study is described below. For all microarray experiments, each measurement point is the average of two biological replicates, each profiled as a technical replicate in dye-swap, yielding four replicates that were averaged and statistically analyzed by Limma (Smyth, ) versus either wild-type or wild-type at t = 0. All procedures were identical and are described in detail in Kemmeren et al (), with protocols submitted to ArrayExpress. Calculations were made using the statistical language R version 3.0.1 on a Linux machine running CentOS 5.5, with packages and scripts provided in the Supplementary R Packages. The expression data (Miller et al, ) (Fig E) was obtained from ArrayExpress (accession number E-MTAB-439) and normalized with rma from the R package affy. For this data, expression changes are the log2-ratios relative to the median of the four t = 0 wild-types. [...] The Van Dijk et al () data (Fig F) were obtained from Sequence Read Archive (accession number SRP005955). Reads were mapped with bowtie (Langmead et al, ) to the yeast genome R64.1.1 with standard settings. Read counts per gene were calculated with featureCounts from the R package Rsubread (Liao et al, ). Normalization and log ratios were obtained using the R package edgeR (Robinson et al, ). […]

Pipeline specifications

Software tools Bowtie, Subread, edgeR
Databases SRA
Application RNA-seq analysis