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Sample GSM3043082 Query DataSets for GSM3043082
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.
 
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
 
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

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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