description: Protocols: 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 30°C. The resulting colonies were re-plated on quadruple drop-out SD plates with 40 μg/ml X-α-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 3-5). 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 4-7 and final results are summarized in Table S1, sheet 2. Sheet 3 includes locations found in screens with more than three different baits.
attributes.tag: ENA first public
attributes.value: 2022-01-14
attributes.tag: ENA last update
attributes.value: 2022-01-14
attributes.tag: External Id
attributes.value: SAMEA12110017
attributes.tag: INSDC center alias
attributes.value: ZMBH - University of Heidelberg
attributes.tag: INSDC center name
attributes.value: ZMBH - University of Heidelberg
attributes.tag: INSDC first public
attributes.value: 2022-01-14T12:16:19Z
attributes.tag: INSDC last update
attributes.value: 2022-01-14T12:16:19Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-4946:Y2H library interactions 2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: plasmid transfected into yeast grown in SD agar
attributes.tag: organism part
attributes.value: plasmid
attributes.tag: replicate
attributes.value: 2
attributes.tag: sample name
attributes.value: E-MTAB-4946:Y2H library interactions 2
attributes.tag: strain
attributes.value: 927