Computational protocol: Nuclear RNA Sequencing of the Mouse Erythroid Cell Transcriptome

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Protocol publication

[…] Library preparation was performed according to Illumina PE genomic protocol, incorporating improvements suggested in Quail et al 2008, with all reactions scaled according to starting DNA quantity , . Using the Illumina GA-IIx platform, we sequenced paired-end 36 bp reads from the generated libraries. Sequencing data was submitted to the Sequence Read Archive (SRA, http://www.ebi.ac.uk/ena/data/view/ERP000702). Sequences were aligned using Bowtie , by suppressing alignments to only 1 best reportable alignment with a maximum number of 2 mismatches within 28 nucleotides of seed length in the high quality end. A gap width of 2500 bp was allowed between paired end reads. When comparing to G1E and G1E-ER4+E2 RNA-Seq data all reads were mapped as single end reads using the indicated Bowtie settings. Sequences were visualised using SeqMonk and the UCSC genome browser. We used the mouse Ensembl gene annotations throughout (genome version NCBIM37). Genes smaller than 300 bp were excluded from the list of genes investigated in the RNAPII stalling section. Peaks were identified using SISSRs (p<0.001) and SeqMonk . Perl, Java and R were used for further data processing. SPSS (version 18) was used for statistical analysis as detailed in the text. […]

Pipeline specifications

Software tools Bowtie, SeqMonk, SISSRs
Databases UCSC Genome Browser
Applications RNA-seq analysis, Genome data visualization
Organisms Mus musculus, Homo sapiens