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| Status |
Public on May 25, 2022 |
| Title |
ChIPseq IP WT Rep D |
| Sample type |
SRA |
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| Source name |
whole cell lysate and immunoprecipitated DNA
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| Organism |
Cereibacter sphaeroides 2.4.1 |
| Characteristics |
genotype: wild-type
oxygen: aerobic
growth phase: mid-log growth phase
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| Treatment protocol |
cells fixed and crosslinking was performed with 10mM sodium phosphate buffer, 1% formaldehyde on ice for 5 minutes before stopping with 100mM glycine.
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| Growth protocol |
500mL of Sistrom's minimal media, sparged with 69% N2, 30% O2, 1% CO2 for aerobic growth to an OD ~ 0.5 mid-log
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells lysed and DNA sheared with sonication, DNA-protein fragments trimmed with micrococal nuclease and Rnase treated, Antibody coimmunoprecipitation, sepharose bead pull down, 250mM LiCl wash, 600mM NaCl wash, 300mM NaCl wash, TE buffer wash, protein-DNA complexes eluted from beads, crosslinking revered by heating, DNA purified with QIAquick PCR purification Kit
ChIP-seq library preparation and sequencing was performed at GeneWiz. Libraries for sequencing were created using the Illumina TruSeq ChIP library preparation kit (Illumina) following the standard protocol. ChIP-seq libraries were sequenced on an Illumina HiSeq 3000 2x150bp reads using the standard protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
ChIP-seq analysis
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| Data processing |
For RNA-seq analysis, the paired-end FASTQ files were split into R1 and R2 files and R1 files were retained for further analysis as previous data contained only single-end reads. Reads were trimmed using Trimmomatic version 0.3 with the default settings except for a HEADCROP of 5, LEADING of 3, TRAILING of 3, SLIDINGWINDOW of 3:30, and MINLEN of 36. After trimming the reads were aligned to the R. sphaeroides 2.4.1 genome sequence (GenBank accession GCA_000012905.2) using Bowtie2 version 2.2.2 with default settings except the number of mismatches was set to 1. Aligned reads were mapped to gene locations using HTSeq version 0.6.0 using default settings except for the “reverse” strandedness argument was used. edgeR version 3.26.8 was used to identify significantly differentially expressed genes from pairwise analyses, using Benjamini and Hochberg false discovery rate (FDR) less than 0.05 as a significance threshold. Raw sequencing reads were normalized using the reads per kilobase per million mapped reads (RPKM).
For ChIP-seq analysis, the paired-end FASTQ files were trimmed with Trimmomatic version 0.3 with default settings except for LEADING:3, TRAILING:3, SLIDINGWINDOW:3:30, and MINLEN:36. The trimmed paired-end reads were aligned to the R. sphaeroides 2.4.1 genome sequence (GenBank accession GCA_000012905.2) using Bowtie2 version 2.2.2 with default settings except the number of mismatches was set to 1. Picard-tools version 1.98 and Samtools version 1.2 were used to clean, sort, and index the alignment file using default settings. Areas of enrichment of IP over INPUT (peaks) were identified using MOSAiCS version 2.28 using two factor analysis and an analysisType of “IO”. Conversion of BAM to ELAND format for MOSAiCS was performed using Pyicos version 2.0.6 and default settings. WIG files were generated for each sample using QuEST version 2.4 and default settings.
Genome_build: GCF_000012905.2
Supplementary_files_format_and_content: Normalized RPKM read counts for RNA-seq. Raw read counts from HTSeq for RNA-seq. WIG files for ChIP-seq
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| Submission date |
Oct 26, 2021 |
| Last update date |
May 27, 2022 |
| Contact name |
Kevin S Myers |
| E-mail(s) |
kmyers2@wisc.edu
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| Organization name |
University of Wisconsin - Madison
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| Department |
Great Lakes Bioenergy Research Center
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| Street address |
5127 WEI, 1552 University Ave
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| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53726 |
| Country |
USA |
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| Platform ID |
GPL30899 |
| Series (1) |
| GSE186600 |
The Essential Rhodobacter sphaeroides RSP1056-RSP0847 (CenKR) Two-Component System Regulated Tol-Pal and Cell Envelope Biosynthesis |
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| Relations |
| BioSample |
SAMN22570641 |
| SRA |
SRX12777925 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5657415_IP_WT_Rep4.wig.gz |
14.0 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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