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| Status |
Public on Feb 02, 2021 |
| Title |
UTA39 (∆xrrA) Rep 3 |
| Sample type |
SRA |
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| Source name |
B. anthracis cell culture
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| Organism |
Bacillus anthracis |
| Characteristics |
strain: Ames ancestor
genotype: xrrA-null
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| Growth protocol |
B. anthracis strains were cultured in 25 ml of casamino acids medium supplemented with 0.1% w/v of glucose at a starting OD600 of 0.08 in 5% atmospheric CO2 (CA-CO2). All cultures were incubated with shaking at 200 r.p.m.
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| Extracted molecule |
total RNA |
| Extraction protocol |
At early stationary phase (OD600 = 1.0 – 1.5), cells from 10-ml samples were centrifuged at 10,000 x g for 15 min. Total RNA was extracted using two consecutive treatments of saturated acid phenol (pH 4.3), followed by treatment with chloroform. To precipitate the RNA from the final aqueous phase, 20 ng of glycogen, one-tenth volume of 3 M sodium acetate, and 3 volumes of 100% ice-cold ethanol were added to the samples. RNA was precipitated at -80◦C for one hour, followed by centrifugation. The resulting RNA pellet was washed twice with ice-cold 75% ethanol before resuspending in DEPC-treated sterile water.
RNA libraries were prepared for sequencing using standard Illumina protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
Two paired-end sequencing runs were performed
UTA37vsUTA39_CuffDiff.txt
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| Data processing |
Illumina RTA v2 software was used for base calling.
The quality of the reads was assessed using FastQC (Andrews, 2010).
Low quality bases and Illumina Adapter sequences were removed using Trim Galore! Galaxy Version 0.6.3 (Krueger, 2012).
A second round of FastQC analysis was done to verify the removal of low-quality bases and Illumina Adapter sequences.
Trimmed reads were aligned to the complete Ames Ancestor genome (NCBI accession numbers AE017334.2 for chromosome, AE017336.2 for pXO1, and AE017335.3 for pXO2) using the GCF_000008445.1_ASM844v1_genomic.fna FASTA file obtained from the NCBI website. Bowtie2 Galaxy Version 2.3.4.3 (Langmead & Salzberg, 2012) with default parameters was used to align the paired-end reads to the genome.
BAM files obtained from mapped reads from each of the two sequencing runs were pooled using Convert, Merge, Randomize Galaxy Version 2.4.0.0 (Barnett et al., 2011), resulting in a single BAM file per triplicate per strain.
The Cufflinks/Cuffcompare/CuffDiff pipeline (Trapnell et al., 2010) was used for transcript assembly using the GCF_000008445.1_ASM844v1_genomic.gff reference annotation file from NCBI, count the number of reads mapped to each annotated transcript, and calculate differential transcript expression between the strains. The Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values per assembled transcript were obtained and differential expression was calculated as the log2 (fold-change) in FPKM values between the parent strain and each sRNA-null strain, with an adjusted p-value cut-off for significance of 0.01.
BamCoverage Galaxy Version 3.3.2.0.0 (Ramírez et al., 2016) was used to create BigWig files for visualization of read coverage over the genome on Integrative Genomics Viewer (IGV) (Thorvaldsdóttir et al., 2013).
Genome_build: GCF_000008445.1_ASM844v1
Supplementary_files_format_and_content: Tab-delimited text files showing log2(fold-change) in expression between the parent strain and each sRNA mutant.
Supplementary_files_format_and_content: BigWig files showing read coverage across the genome per DNA strand (F and R) per strain.
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| Submission date |
Jun 12, 2020 |
| Last update date |
Feb 03, 2021 |
| Contact name |
Theresa Marie Koehler |
| E-mail(s) |
Theresa.M.Koehler@uth.tmc.edu
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| Organization name |
McGovern Medical School
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| Department |
Microbiology and Molecular Genetics
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| Street address |
6431 Fannin Street, MSB 1.508
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| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77054 |
| Country |
USA |
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| Platform ID |
GPL28686 |
| Series (1) |
| GSE152356 |
Plasmid-encoded Small Regulatory RNAs Regulate Chromosome Gene Expression in Bacillus anthracis |
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| Relations |
| BioSample |
SAMN15223271 |
| SRA |
SRX8537467 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4613414_UTA39_Rep3_F.bigwig |
10.5 Mb |
(ftp)(http) |
BIGWIG |
| GSM4613414_UTA39_Rep3_R.bigwig |
10.7 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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