Illumina HiSeq 2000 sequencing; Interactions of CAF1-NOT complex components from Trypanosoma brucei
Y2H library - Random fragments of Trypanosoma brucei genome The samples were applied from a Y2H libraries using primers annealing up and downstream of the cloned sequence. The PCR product were send to sequence to identify the putative interactions. The Matchmaker GAL4 Two-Hybrid System3 (Clontech) was used, with Gateway cloning of amplified open reading frames to create bait plasmids. The trypanosome prey library, sequencing methods and bioinformatic analysis have been previously described15. Briefly, bait plasmids were transformed into AH109 yeast, and the pool of prey plasmids were transformed into the Y187 strains. The cells were allowed to mate, plated on SD agar plates lacking tryptophan, leucine, or both (double drop-out medium) to calculate mating efficiency, then plated without tryptophan, leucine, adenine and histidine (quadruple drop-out medium), and incubated for 3 to 5 days at 30C. The resulting colonies were re-plated on quadruple drop-out SD plates with 40 g/ml X--Gal and 3-amino triazole (3-AT, 0.5 to 2mM), and incubated for 3 to 5 days. Blue colonies were then pooled and grown overnight in quadruple drop-out medium with 3-amino triazole for plasmid isolation. The plasmid inserts were amplified with bar-coded primers, pooled and sent for library preparation by David Ibbersson (BioQuant, Heidelberg, Germany), then sequenced (Illumina Hi-Seq, EMBL, Heidelberg., Germany). The numbers of reads obtained are in Table S1, Sheet 1. Reads were aligned to the T. brucei 927 genome, as described15. We selected sequences that were present at least 10 times, and that had annotated open reading frames that were in frame with the DNA-binding domain (Table S1, sheets 35). We then chose a list of unique open reading frames with only one copy each of repeated genes22. We also restricted the selection to open reading frames that included no more than 13 codons upstream of the start codon, and included at least 6 codons before the stop codon. Individual results are in Table S1, sheets 47 and final results are summarized in Table S1, sheet 2. Sheet 3 includes locations found in screens with more than three different baits.