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| Status |
Public on Sep 20, 2019 |
| Title |
rep2 50mM MgSO4 gate 7 [degenerate_library_sort_seq] |
| Sample type |
SRA |
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| Source name |
rep2 50mM MgSO4 gate 7
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
replicate: replicate 2
agent: 50mM MgSO4
facs gate: 7
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| Treatment protocol |
After sorint, before plasmid extraction, E. coli libraries were grown overnight in 2xYT media [degenerate_library_sort_seq]
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| Growth protocol |
Cells were grown in the indicated conditons for 6hr, then sorted into consecutive bins based on their YFP fluorescence. E. coli libraries were grown overnight in 2xYT media [degenerate_library_sort_seq]
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
plasmid DNA was extracted with the Quiagen plasmid Miniprep Kit [degenerate_library_sort_seq]
For the two mutagenized regions of the plasmid to be brought into close enough proximity (< 790 bp) for paired-end Illumina sequencing, plasmids were digested with XhoI and then self-ligated (T4 ligase, 4 hr). To isolate only self-ligation products, and not cross-ligation products, ligation reactions were cleaned (Zymo PCR Clean Up) and gel purified to select for the correct size on FlashGels (Lonza). Two PCR reactions were performed, both using KAPA HiFi Hotstart, to add Illumina sequencing adaptors and barcodes. First, ligation reaction products were amplified for 30 cycles (95 °C for 30 s, 65 °C for 15 s, 72 °C for 120 s) with primers CJM642 and CJM643 in an emulsion PCR (Micellula Emulsion PCR) to avoid PCR chimeras. Second, purified PCR product from the first reaction was subjected to a second PCR with barcoding primers for 9 cycles (95 °C for 30 s, 65 °C for 15 s, 72 °C for 60 s). Final products were quantified (NanoDrop), normalized, combined, and sequenced on an Illumina NextSeq. [degenerate_library_sort_seq]
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
plasmid DNA
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| Data processing |
Library strategy: PCR amplicon sequencing [degenerate_library_sort_seq]
reads with errors outside of the variable/barcode and mutated nucleotides were discarded to remove low quality reads
The frequency of each mutant in each bin was calculated as a function of the total reads in each bin and the total number of cells sorted into each bin (McClune et al., 2018)
Variants with fewer than 25 total reads were discarded
Gaussian functions were fit to each distribution (in log10YFP units), from both the ON and OFF sorts (SciPy optimize package).
The standard deviation error on the estimated log(YFP) mean was used as a metric to filter poorly fit sequences (sequences were removed if ONstdev + OFFstdev > 2)
Fold induction was calculated as the ratio of the fit means between the induced (10uM MgSO4) and uninduced (50mM MgSO4) states.
Supplementary_files_format_and_content: csv file. First line contains column titles
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| Submission date |
Oct 03, 2018 |
| Last update date |
Sep 20, 2019 |
| Contact name |
Conor James McClune |
| E-mail(s) |
conor.mcclune@gmail.com
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| Organization name |
Massachusetts Institute of Technology
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| Department |
Biology
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| Lab |
Michael Laub
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| Street address |
31 Ames St
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL21117 |
| Series (2) |
| GSE120780 |
Engineering orthogonal signaling pathways reveals the sparse distribtion of protein protein interactions in sequence space [degenerate_library_sort_seq] |
| GSE120789 |
Engineering orthogonal signaling pathways reveals the sparse distribtion of protein protein interactions in sequence space |
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| Relations |
| BioSample |
SAMN10171438 |
| SRA |
SRX4793059 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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