iptg concentration (μm): 10 3-at concentration (mm): 2 duration of experiment (hours): 18 media: agarose gel
Extracted molecule
other
Extraction protocol
After each selection, all of the cells were scraped off the plate. The randomized portion of the prey vector containing the selected transcription factor binding sites were extracted and amplified using PCR. The PCR products from different experiments were digested with different restriction enzymes in the first step of uniquely encoding sequences from different experiments. The restriction enzyme fragments were then barcoded, pooled and sequenced using a Illumina Genome Analyzer IIx machine. See the supplemental material of Noyes et al. (2008). Cell. 133(7):1277-1289 for a more detailed description of the experimental protocol employed for each selection.
Library strategy
OTHER
Library source
synthetic
Library selection
other
Instrument model
Illumina Genome Analyzer IIx
Data processing
The sequences from each B1H experiment were labeled using a 3bp barcode. The raw reads were parsed and assigned to the appropriate data set, based on the barcode sequence. Furthermore, only reads that matched the expected constant region, TGTACCGCNNNNNNATTTTTNNNNTGGCCAGCT, (N's indicate randomized positions) , except for up to two mismatches at non-randomized positions, were assigned to the appropriate data sets. All of the reads that did not meet these criteria were placed in the 'unassigned' set. Reads with randomized regions that included an 'N' character were also placed in the 'unassigned' set. The *.uniqSeq_2_counts.tsv files indicate the number of times each unique 6bp long randomized binding site was observed. The single liquid media experiment, 4hrs_50μM_IPTG_5mM_3-AT_liquid, was sequenced in a different lane together with other multiplexed experiments. It's barcode was GAT. The 6bp upstream of the constant region ATTTTT were parsed out and used to generate the corresponding *.uniqSeq_2_counts.tsv file.