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Pipeline publication

[…] rand Synthesis Module from New England Biolabs, Inc. After DNA shearing by ultrasonication (Covaris S2) and treatment with S1 nuclease (New England Biolabs, Inc.), samples were subjected to standard Illumina fragment library preparation using indexed adaptors. Resulting libraries were pooled in equimolar quantities for 75-bp single-read sequencing on Illumina HiSeq 2000 and distributed on several lanes, resulting in ∼30–90 million reads per sample., Alignment of the short reads to the mm9 transcriptome was performed with pBWA software, and a table of read counts per gene was created based on the overlap of the uniquely mapped reads with the Ensembl Genes annotation version 61 for mm9, using BEDtools (version 2.11; ). The raw read counts were then normalized with the DESeq R package (version 1.8.1; ), and the sample to sample Euclidean distance was computed based on the normalized counts to explore sample to sample correlation. After normalization, testing for differential expression was performed with DESeq, and accepting a maximum of 10% false discoveries (10% FDR) resulted in 96 up-regulated and 48 down-regulated genes in Kitint HSCs. For SCF trigger experiments, DEGs between Kitint versus Kitint + SCF and Kithi versus Kithi + SCF were compared, and DEGs unique to Kitint + SCF versus Kitint + SCF or DEGs common to both gene lists were identified. To identify enrichment for particular biological processes associated with the DEGs, the DAVID GO/BP/FAT database () was used. Enrichment scores were calculated (−log transformation of the DAVID EASE score) to determine overrepresentation of particular biological processes. To quantify gene expression levels, an expression score defined as the median of all normalized counts of the DEGs associated with that particular GO term was calculated. Subsequently, expression scores for the top five GO biological process terms across the four experimental conditions were depicted in a heat map., We thank S. Piontek and S. Böhme for expert technical assistance and V. Grinenko and B. Wielockx for discussion., C. Waskow is supported by the Center for Regenerative Therapies Dresden, by the Deutsche Forschungsgemeinschaft (DFG) Sonderforschungsbereich (SFB) 655 (B9), SFB 127 (A3), and FOR203 […]

Pipeline specifications

Software tools BEDTools, DESeq, DAVID
Organisms Mus musculus