Computational protocol: Mechanisms That Determine the Internal Environment of the Developing Brain: A Transcriptomic, Functional and Ultrastructural Approach

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Protocol publication

[…] Short reads were trimmed to remove ambiguous bases from the start and segments with low quality scores from the end, as indicated by the ascii character “B” in Illumina 1.5 phred score encoding. Trimmed reads were mapped with Bowtie version 0.12.7 to the Ensembl rat genome, release 61 . Reads that did not map uniquely were discarded. The number of reads mapped to nuclear genes was determined with HTSeq version 0.4.7p4, using the default “union” counting option. Differential expression between the adult and embryonic samples was detected using an exact test in the Bioconductor edgeR package, version 2.4.0 , with common dispersion used to estimate variance between samples. Genes considered significantly differentially expressed were those with a p-value of less than 0.05 after Benjamini-Hochberg false discovery rate correction. A combination of gene ontology annotation and manual curation was used to select genes encoding proteins that form part of a cell junction. Gene ontology descriptions for rat were downloaded from Biomart , and genes with “junction” mentioned in their gene ontology description were selected. Junction genes of interest were then extracted from this list. Similar searches were carried for other functional categories as described in the Results/Discussion below. For initial analysis genes with >100 sequence reads and age-related fold changes (FC) >2.0 (log2FC >1.0) were collated and are summarised in , , , . For more specific analysis of some particular function categories a lower cut-off of 10 sequence reads was used. Illumina RNA sequencing data have been deposited with the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession code GSE44072. […]

Pipeline specifications

Software tools Bowtie, HTSeq, edgeR
Application RNA-seq analysis
Organisms Rattus norvegicus, Caenorhabditis elegans