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| Status |
Public on Jun 01, 2015 |
| Title |
Sample_4A-M1T1_DPTP |
| Sample type |
SRA |
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| Source name |
Late-log phase culture
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| Organism |
Streptococcus pyogenes |
| Characteristics |
strain: M1T15448
genotype/variation: M1T1DPTP
phenotype: Lacking -SP-PTP
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| Treatment protocol |
Using pFW6 vector, S. pyogenes mutants lacking SP-PTP were derived. The washed concentrated bacterial pellet suspension was treated with phage-lysin to lyse the bacterial cells.
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| Growth protocol |
All cultures were grown in Todd Hewitt broth supplemented with 0.5% yeast extract. Cultures were grown at 37oC till O.D. reached at 0.8. (wt) mutant (0.65) i.e. till late log phase. The cultures were then centrifuges at 4oC and washed once with sterile autoclaved PBS. The cell were then resuspended in PBS in the 1/50th of the starting culture volume.
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| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted using Norgen RNA isolation kit (canada).
The high quality total RNA samples were obtained by DNase treatment. The samples were then sent to BGI Americas. rRNA was removed from the total RNA samples.cDNA was then made using mRNA as template. Short fragments were made by heat treatment amd the shortfragment were purifiedand resolved with EB buffer for endreparation and single nucleotide A ( adenine) addition. Short fragments were then connected to adapters. Subsequently, folowwing agarose gel electrophoresis, the suitable fragments were selected for the PCR amplification. All qc steps i.e. for quatification and quality of sample library ABI bioanalyzer and ABI StepOnePlus realtime PCR system were used. Library were sequenced using IlluminaHiseq 2000.
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Primary sequencing data that produced by Illumina Hiseq 2000, called as raw reads were subjected to quality control to determine if resequencing step is needed. After QC, raw reads were filtered into clean reads which was then aligned to reference sequence Streptococcus pyogenes MGAS5005 NC_007297.1 with SOAPaligner/SOAP2 v2.20 [Li. R, Yu,C., LiY et al Bioinformatics :Doi:10.1093/bioinformatics/btp336. 259(15)1966-1967]. The alignment data were utilized to calculate distribution of reads on reference genes and perform coverage analysis. Upon QC pass, down stream analysis including gene expression, novel transcript prediction and annotation were done. Gene ontology enrichment analysis were used for pathwat enrichment analysis. In order to assess the effectiveness of selected mapping tool, the comparative analysis between SOAPAligner/SOAP2 and other popular-alignment tool BOWTIE [Langmead B. et al (2009) genome biology 10:25-34/ http://Bowtie-bio.sourceforge.net/index.shtml]. Because the SOAPaligner/SOAP2 performed better, quantification analysis of gene expression was done with SOAPaligner/SOAP2. For significance of didgital gene expression profile alogorithms as published [Audic et al (1997) Gemome Res. 7:986-985].. We used FDR< 0.001 [benjamini and Yekutieli(2001)Ann Statistics29:1165-1188] and the absolute value Log2 ratio > 1 as the threshold to judge the significance of gene expression difference. KEGG was used to perform Pathway enrichment analysis. For gene refinement, transcripts reads were assemble by using CUFFLINK software [Roberts et al Bioinformatics: doi:10.1093/bioinformatics. btr355].
Genome_build: NC_007297.1
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| Submission date |
Jun 11, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Vijay Pancholi |
| E-mail(s) |
[email protected]
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| Phone |
614-688-8053
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| Organization name |
Ohio State University
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| Department |
Pathology
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| Lab |
Bacterial Pathogenesis
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| Street address |
420W 12th Ave, TMRF-288
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| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43210 |
| Country |
USA |
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| Platform ID |
GPL18784 |
| Series (1) |
| GSE58412 |
RNA-seq analysis of S. pyogenes SP-PTP mutants |
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| Relations |
| BioSample |
SAMN02850902 |
| SRA |
SRX590719 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1410557_Sample4A_M1T1_DPTP.Gene.rpkm.txt.gz |
27.0 Kb |
(ftp)(http) |
TXT |
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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