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Protocol publication

[…] For comparing freshly isolated tips to freshly isolated stalks (), Fastq files were filtered for low quality reads (TopHat 2.0.6 () guided by RefSeq gene models (UCSC). Raw counts per transcripts were obtained using featureCounts and differentially expressed genes (>2 fold difference) identified using edgeR 2.6.12 (). Hierarchical unsupervised clustering was performed using published foetal lung RNAseq data as a comparison (). Gene Ontology and Panther Pathway analysis was performed in DAVID (, ).To compare the human and mouse embryonic tip transcriptome, we compared our human tip RNA seq with previously published mouse E11.5 tip microarrays (GEO accession numbers: GSM1968996, GSM1968997, GSM1968998, GSM1968999, GSM1969000). We first assessed whether transcripts of orthologous mouse/human genes (defined by the HomoloGene database) were present in each data-set (). Genes were excluded which had RPKM values < 1 (RNAseq) and expression values of <5 (microarray). To estimate the relative levels of these transcripts between the mouse microarray and human RNAseq data, we reasoned that the microarray signal saturates and therefore generated a scatter plot of mean microarray signal, versus mean log-transformed RPKM for each orthologous gene identified in mouse and human ().RNAseq for the cultured organoids was performed on an independent sequencing run. To compare freshly isolated human tip and stalk samples with the cultured organoids, the RUVSeq R package (ruvg using housekeeping genes) was used to control for the batch effect in the data () using hidden factor k = 1. Multi-dimensional scaling plot, heat map and box plots ( and ) were produced in R using the batch-corrected data. […]

Pipeline specifications

Software tools TopHat, Subread, edgeR, DAVID, RUVSeq
Databases HomoloGene
Application RNA-seq analysis
Organisms Homo sapiens, Mus musculus