Similar protocols

Protocol publication

[…] Illumina mRNA-seq libraries were prepared with the TruSeq RNA kit (version 1, Rev A), using 1 μg of total RNA according to the manufacturer’s instructions. For HiSeq. 2000 sequencing, eight libraries were pooled per sequencing lane. The pooled library samples were sequenced on the Illumina Hiseq. 2000. The raw data from the sequencing run is initially assessed for Illumina low Phred base score and the presence of adapter sequences using the FastQC program. The phred score of Q30 was obtained for all the FastQ RNAseq Files. Following initial analysis, reads that passed the QC were processed using the New Tuxedo Algorithm,. Briefly, RNAseq reads were aligned to the Human reference genome hg38 annotation using the HISAT2 algorithm and read counting overlapping gene counting overlapping gene features was done using HTseq. Mapping efficiency and quality were assessed using the RNAseq software. The number of reads mapping to annotated gene features was counted using HTSeq using an hg38 General Feature Format (GTF) annotation file. The resulting counts were imported to R and the Bioconductor package DESeq. 2 was used for differential gene expression analysis.The Nucleotide Sequences are archived at European Nucleotide Archive (ENA) with accession number PRJEB23139 as primary accession number and ERP104872 as secondary accession number. [...] The hierarchical clustering and Venn plot were carried out using rpudplus, hclust and Venn diagram function in R-Studio employing differentially expressed pre-ranked genes which were associated with extracellular secretory function as classified using TopGO R-Studio package and GeneTrail2. The principle component analysis (PCA) based on regularized log transformed FPKM values with an adjusted p-value < 0.05. obtained from DEseq. 2 and was carried out using ggfortify and ggplot2 packages in R-Studio. (R Tutorial with Bayesian Statistics Using OpenBUGS, Chi Yau, ASIN: B006ZP4SKW). […]

Pipeline specifications

Software tools FastQC, Tuxedo, HISAT2, HTSeq, DESeq, Hclust, TopGO, GeneTrail, Ggplot2
Databases ENA
Organisms Mus musculus
Diseases Cicatrix, Inflammation, Wounds and Injuries
Chemicals Calcium