Determination and integration of abundance, processing efficiency, and degradation kinetics of the mRNAs encoding all T. brucei redox and pentose phosphate pathway enzymes

Since over 40 enzymes will be investigated for their mRNA abundance, processing, and degradation kinetics, the less tedious and more accurate Next Generation Sequencing of the entire mRNA repertoire of the cell is employed. To optimise the proportion of useful sequence, while including RNA fragments that are products of of degradation, rRNA is depleted using the eukaryotic Ribominus kit (Ambion). Two biological replicates are treated with Sinefungin and Actinomycin D to inhibit RNA processing and transcription, for different time periods. RNA is collected before treatment, 5 min after the addition of Sinefungin, 5 min after addition of ActD (+Sin), then 10 min after, 20, 30, 60 and 120 min. The samples are sequenced using the Illumina standard protocol, and the sequenced reads are aligned to the reference genome TREU927, and the number of reads aligned to each ORF are determined.


Gene expression in Trypanosoma brucei

Projects: SilicoTryp

Christine Clayton

Experimentalists: Federico Rojas, Abeer Fadda

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Views: 2537

Created: 24th Feb 2012 at 14:51

Last updated: 20th Nov 2014 at 09:34

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