Illumina HiSeq 2000 sequencing; Transcriptome of Trypanosome brucei after PUF2 Knock-down - polysome fractions
Transcriptome of Trypanosome brucei after PUF2 Knock-down - polysome fractions
Culturing and transfection of trypanosomes (Lister 427 strain expressing the tet repressor from pHD1313 [1313 trypanosomes]) were performed as described in reference 35 (see ref in associated publication. doi 10.1128/EC.00018-14). For the expression of tetracycline-inducible genes, 100 ng ml−1 of tetracycline (Tet) (36) was used. For monitoring the growth, procyclic cells were grown in minimal essential medium-Pros medium at 27°C and were diluted to a density of 5 × 105 cells ml−1. RNA interference against PUF2 was done by making a stem-loop RNAi plasmid as described in reference 36. A 432-bp region (bp 399 to 831 of the ORF) of the PUF2 gene was selected for RNAi (the sequence was verified for its specificity by using the RNAit program [http://trypanofan.path.cam.ac.uk/software/RNAit.html]). The stem-loop PUF2 RNAi vector was referred to as pHD2183, and after digestion with NotI the vector was used to transfect 1313 bloodstream- and procyclic-form cells. Approximately 5 × 108 1313 bloodstream-form cells (density, ≈1 × 106 cells ml−1) were fractionated as described in reference 45. Polysomes were purified from the same number of cells according to the method described in reference 46. The polysomal samples were trichloroacetic acid precipitated and resuspended in 4× Laemmli sample buffer. Trifast follow rRNA depletion by RNAse-H treatment For the RNAi experiment, trypanosomes were grown for 1 day in the presence of tetracycline, and poly(A)+ mRNA was sheared and prepared for sequencing according to the standard Illumina protocol. One PUF2 RNAi batch was sequenced on the GAII and the second with HiSeq. Data analysis, including motif searches and enrichment calculations, were performed as described previously (22, 54). All data were log transformed before the correlation coefficient calculations, in order to reduce the influence of outliers and correct for a nonnormal distribution of the data.