|
| Status |
Public on May 25, 2022 |
| Title |
RNAseq D56A Rep B |
| Sample type |
SRA |
| |
|
| Source name |
whole cell lysate and total RNA
|
| Organism |
Cereibacter sphaeroides 2.4.1 |
| Characteristics |
genotype: RSP_0847 D56A
oxygen: aerobic
growth phase: mid-log growth phase
|
| Treatment protocol |
cells fixed with 95% EtOH and 5% acidic phenol:chloroform
|
| 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
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hot acid phenol:chloroform extraction, NaOAc/isopropanol precipitation, ethanol wash, Dnase treatment, Qiagen Rneasy kit clean-up and concentration
RNA-seq library preparation and sequencing was performed at the Joint Genome Institute. Libraries for sequencing were created using the Illumina TruSeq Stranded Total RNA kit (Illumina) following the standard protocol. RNA-seq libraries were sequenced on an Illumina NextSeq in 2x151 reads using the standard protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
RNA-seq analysis
Lakey_RNAseq_Log2RPKM_Values.txt
Lakey_RNAseq_HTseq_RawCountValues.txt
|
| 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
|
| |
|
| Submission date |
Oct 26, 2021 |
| Last update date |
May 25, 2022 |
| Contact name |
Kevin S Myers |
| E-mail(s) |
kmyers2@wisc.edu
|
| Organization name |
University of Wisconsin - Madison
|
| Department |
Great Lakes Bioenergy Research Center
|
| Street address |
5127 WEI, 1552 University Ave
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53726 |
| Country |
USA |
| |
|
| Platform ID |
GPL30898 |
| Series (1) |
| GSE186600 |
The Essential Rhodobacter sphaeroides RSP1056-RSP0847 (CenKR) Two-Component System Regulated Tol-Pal and Cell Envelope Biosynthesis |
|
| Relations |
| BioSample |
SAMN22570668 |
| SRA |
SRX12777884 |
