|
| Status |
Public on Nov 28, 2018 |
| Title |
Lrp::kanR Rich logarithmic rep2-ChIPseq extracted |
| Sample type |
SRA |
| |
|
| Source name |
Culture lysate
|
| Organism |
Escherichia coli |
| Characteristics |
strain: K12 MG1655
genotype: Lrp::kanR
media: MOPS rich defined media + glycerol
time of harvest: logarithmic phase
chip antibody: anti-Lrp monoclonal antibody (NeoClone, custom antibody clone# 1G4D9A10, Lot# 2016E25-001)
|
| Treatment protocol |
Cells were cross-linked by addition of 1% formaldehyde.
|
| Growth protocol |
Cells isolated from a single colony were grown in liquid culture at 37C until harvest.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA was harvested using Qiagen RNAProtect. RNA was isolated, and rRNA depletion was performed according to the Illumina Ribo-Zero rRNA Removal Kit for Bacteria.
Specific Lrp-DNA complexes were isolated using a monoclonal antibody developed against Lrp, and DNA was isolated by phenol-chloroform extraction.
DNA was preprared for sequencing using the NEBNext DNA Library Prep Kit. RNA was prepared fro sequencing using the NEBNext Ultra Directionaly RNA Library Prep Kit.
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| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
KO_RDM_log_E117
RDM_log_called_peaks.bed.gz
RDM_log_actual_10bp_robust_z.bedgraph.gz
RDM_log_actual_10bp_WT_RDM_log_E40_Sub_KO_RDM_log_E117_actual.bedgraph.gz
RDM_log_actual_10bp_WT_RDM_log_E9_Sub_KO_RDM_log_E117_actual.bedgraph.gz
|
| Data processing |
pre-processing RNA-Seq: Sequencing adapters were removed from all sequences using CutAdapt version 1.8.1 (Martin 2011) with parameters --quality-base=33 -a AGATCGGAAGAGC -A AGATCGGAAGAGC -n 3 -m 20 --mask-adapter --match-read-wildcards. Low quality reads were trimmed with Trimmomatic version 0.32 (Bolger et al. 2014) using the parameters LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:20. For all samples, remaining ribosomal reads were removed from analysis (details in the publication).
Wald Test RNA-seq: Gene-centric quantification of RNA expression for all samples was performed using kallisto version 0.43.0 (Bray et al. 2016) with the arguments: quant -t 4 -b 100 --rf-stranded. wald_test.csv files represent a Wald test calculated using sleuth (Pimento et al. 2017) performed on the genotype term of a simple model where transcript_abundance ~ genotype for each condition and time point combination seperately. The lrp::kanR genotype is considered the baseline in this model.
TPM estimates RNA-seq: log2(WT/KO) TPM ratios were calculated from the TPM estimates given by kallisto run as described in the Wald Test RNA-seq step above. Percentile-based 95% confidence intervals are estimated from 100 bootstrap replicates of the log2(WT/KO) TPM ratios (details in publication)
pre-processing ChIP-Seq: Sequencing adapters were removed from all sequences using CutAdapt version 1.8.1 (Martin 2011) with parameters -a AGATCGGAAGAGC -A AGATCGGAAGAGC -n 3 -m 20 --mask-adapter --match-read-wildcards. Low quality reads were trimmed with Trimmomatic version 0.32 (Bolger et al.2014) using the parameters TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:20.
Lrp WT - lrp::kanR ChIP-signal: Median-normalized WT log2(Extracted/Input) - KO(Extracted/Input) signal at every 10 bp is reported for each combination of WT and KO replicates for each time and condition. (details in publication)
Robust-Z ChIP summary signal: Each subtracted replicate in the step above was converted to a Robust-Z score and average between replicates for each time and condition combination to give a final summary signal (details in publication)
Lrp ChIP peaks: High-confidence Lrp binding regions were called through the use of three filters: 1. technical reproducibility estimated by bootstrap replicates sampled from raw reads. 2. biological signal estimated from the Lrp WT- lrp::kanR ChIP signal. 3. Biological reproducibility estimated from the IDR (Li et al. 2011) between biological replicates. (details in publication)
Genome_build: E.coli MG1655 U00096.2 modified to match ATCC47076 strain as reported by Freddolino et al. 2012. All genome annotations were taken from RegulonDB version 9.4 (Gama-Castro S et al. 2016) with genome coordinates converted appropriately
Supplementary_files_format_and_content: Files ending in wald_test.csv: Results of Wald test as described in the Wald Test RNA-seq data processing step for each time and condition combination in the file name. Files are in csv format
Files ending in log2_wt_ko_ratio.tsv: Results of the log2(WT/KO) expression estimates. RNA-seq data processing step for each time and condition combination in the file name. Files are in tab seperated format (tsv). Column 1 is the estimate. Columns 2 and 3 represent a conservative percentile based 95% confidence interval.
Files ending in actual.bedgraph.gz: Results of Lrp WT- lrp::kanR ChIP-signal data processing step for the appropriate combination of WT and lrp::kanR replicates in the file name. Lines include a one 1bp entry for each location reported. Locations resulting in a NaN, inf, or -inf were not included in each .bedgraph
Files ending in robust_z.bedgraph.gz: Results of the Robust-Z ChIP summary signal data processing step for the appropriate combination of time and condition in the file name. Lines include a one 1bp entry for each location reported. Locations resulting in a NaN, inf, or -inf were not included in each .bedgraph
Files ending in peaks.bed.gz: Results of the Lrp ChIP peaks data processing step for each time and condition combination in the file name. Peaks are in .bed6+4 format with ATCC47076 as the chromosome name. The score (fifth) field is the maximum robustZ score found in the peak.The additional 4 fields include: 7. Maximum -log10(qvalue) for the technical filter 8. maximum -log10(qvalue) for the biological filter and 9. maximum -log10(qvalue) for the idr reproducibility filter. 10. peak 0-based offset from start where maximum robustZ score occured. In all cases where -log10(qvalue) resulted in an +infinite value (i.e. when qvalue = 0) the -log10(qvalue) was truncated to the next highest finite -log10(qvalue) in the dataset for that filter.
Files ending in abundance.tsv: Results of quantification of each individual RNA sample from kallisto
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| |
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| Submission date |
Mar 15, 2018 |
| Last update date |
Nov 28, 2018 |
| Contact name |
Peter Freddolino |
| E-mail(s) |
petefred@umich.edu
|
| Organization name |
University of Michigan
|
| Department |
Biological Chemistry
|
| Lab |
Freddolino
|
| Street address |
1150 W. Medical Center Dr.
|
| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48105 |
| Country |
USA |
| |
|
| Platform ID |
GPL21222 |
| Series (1) |
| GSE111874 |
Escherichia coli Lrp regulates one-third of the genome via direct, cooperative, and indirect paths |
|
| Relations |
| BioSample |
SAMN08716833 |
| SRA |
SRX3796912 |
