normalization data transformation protocol
Raw reads were mapped to annotated coding sequences (CDS) from Trypanosoma brucei TREU927 (TriTrypDB v2.4) using Bowtie v0.12.7, with the following parameters: --best --strata -k 2 -m 1 -n 2. Mapped read counts were normalized to the length of each CDS to obtain RPKM. RPKM values were normalized in each sample by dividing by the median of the sample. Ratio of TAP vs. input for each transcript was calculated by adding a pseudocount of 0.1 to each normalized RPKM value prior to division. Genome_build: TbruceiTreu927AnnotatedCDS_TriTrypDB-2.4; Genome_build: ASM244v1 (Trypanosoma brucei strain 927/4 GUTat10.1) Supplementary_files_format_and_content: Files are tab-delimited. The first column represents the gene IDs (according to TriTrypDB). The second column represents median-normalized RPKM values.
nucleic acid library construction protocol
Tandem affinity purification was performed as described before (Panigrahi et al., http://www.ncbi.nlm.nih.gov/pubmed/12649499). Total RNA or RNA associated with Tb927.8.6650 was extracted using TRIzol Reagent (Invitrogen) as per manufacturers’ intsructions. RNA quality was examined using Agilent 2100 Bioanalyzer.
Transgenic PF T. brucei cells harbouring tetracycline-inducible constructs were grown in SDM-79 medium in 27 °C. Cells were seeded at 1.3E+6 cells/ml and harvested 48h after tet-induction at a density of 3.0E+7 cells/ml.
sample treatment protocol
The cells were treated with 100ng/ml tetracycline, for a duration of 48h.