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| Status |
Public on Oct 01, 2011 |
| Title |
tsb_total_RNA |
| Sample type |
SRA |
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| Source name |
P. gingivalis cells
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| Organism |
Porphyromonas gingivalis W83 |
| Characteristics |
medium: TSB
harvest time: mid-log phase
strain: W83
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| Growth protocol |
P. gingivalis strain W83 was routinely maintained on the blood agar plates BAPHK containing defibrinated sheep blood (5% v/v), hemin (1µg/ml), and vitK/menadione (0.5µg/ml), at 37°C in an anaerobic chamber containing 95% nitrogen and 5% carbon dioxide. Cells were cultured under the same conditions in three different media: i) BAPHK; ii) TSB: trypticase soy broth supplemented with hemin (1µg/ml) and vitK/menadione (0.5µg/ml); and iii) MIN: a chemically defined minimal liquid medium providing alpha-ketogluterate and bovine serum albumin (BSA) as the only protein sources. For BAPHK cultures, colonies grown on the plates for 48 hours were used. For liquid cultures, pre-warmed and pre-reduced medium was inoculated with an aliquot of overnight TSB culture so that the dilution rates were 1:100 and 2:100 for TSB and MIN, respectively. Liquid cultures were allowed to reach mid-log phase (OD550 0.4-0.5) prior to harvest.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Lysis of pelleted cells was performed using the MasterPure RNA Purification Kit (Epicentre, Madison, WI, USA). The lysate was treated with Proteinase K at 65°C for 15 min and then filter purified applying the mirVana miRNA Isolation Kit (Applied Biosystems/Ambion, Austin, TX, USA) according to the recommended protocol. To remove genomic DNA, the RNA extract was treated twice with Turbo DNase (Applied Biosystems/Ambion) at 37°C for 30 min, and purified again with the mirVana miRNA Isolation Kit. All samples were subjected to similar procedures except from the purifications steps where the mirVana filter based method was exchanged for MasterPure based RNA purification when preparing the MIN samples. The resulting total RNA samples were subject to partial removal of 16S and 23S RNAs applying the MICROBExpress Bacterial mRNA Enrichment Kit (Applied Biosystems/Ambion) and then fragmented with RNA Fragmentation Reagents (Applied Biosystems/Ambion) followed by ethanol precipitation. Antarctic Phosphatase (New England Biolabs, Cambridge, MA) was used, 5 U in a 20 ul reaction incubated at 37°C for 30 min, to remove 5' phosphate group from the RNA. Fragmented RNA was then subjected to size selection in a 15% Novex TBE-urea polyacrylamide gel (Invitrogen, Carlsbad, CA, USA), eluted, and ethanol precipitated prior to ligation of the 3' oligonucleotide adapter (5'-UCGUAUGCCGUCUUCUGCUUGUidT-3'). The adapter ligation was performed using T4 RNA ligase in 10% DMSO, incubated at 20°C for 6 hrs. The 3' adapter ligated RNA was purified on a 15% Novex TBE-urea polyacrylamide gel (Invitrogen), eluted, ethanol precipitated, and then phosphorylated in a 50 ul reaction containing 40 U T4 Polynucleotide Kinase (New England Biolabs) and 1 mM ATP (Epicentre) for 1h at 37°C. After phenol: chloroform purification and ethanol precipitation, the 5' oligonucleotide adapter (5'-GUUCAGAGUUCUACAGUCCGACGAUC-3') was ligated to the phosphorylated RNA under conditions similar to the 3' adapter ligation. The adapter ligated RNA was gel-purified and reverse transcribed using a primer specific to the 3' adapter (5'-CAAGCAGAAGACGGCATACGA-3') followed by PCR (15 cycles) using the reverse (5'-AATGATACGGCGACCACCGACAGGTTCAGAG TTCTACAGTCCGA-3') and forward (5'-CAAGCAGAAGACGGCATACGA-3') primers. The entire amplified product was size separated, ethanol precipitated, and dissolved in 10 ul resuspension buffer (Illumina) before the samples were subjected to sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Pileup forward: The Illumina sequence reads were mapped to the reference genome Porphyromonas gingivalis W83 (Accession No. NC_002950.2) using the software PerM allowing a maximum of two mismatches. Reads that did not align to the genome were trimmed from the 3' end, one base at a time, and the mapping-trimming process was repeated until the read was aligned or the trimmed length reached 24nt in length. Reads that were not aligned after being trimmed to 24-nt were discarded. The aligned results were separated into forward and reverse-complement groups and converted to the 'pileup' format by SAMtools. This process generated the reads that were mapped to the forward strand of the reference genome
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| Submission date |
Jul 06, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Tsute Chen |
| E-mail(s) |
[email protected]
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| Phone |
617-892-8359
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| Organization name |
The Forsyth Institute
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| Department |
Microbiology
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| Lab |
Chen
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| Street address |
245 First Street
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL13827 |
| Series (1) |
| GSE30452 |
Strand-specific transcriptome profiles of the oral pathogen Porphyromonas gingivalis using genomic tiling microarray and RNA sequencing |
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| Relations |
| SRA |
SRX082173 |
| BioSample |
SAMN00632336 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM755209_tsb_fw.pileup.txt.gz |
237.0 Mb |
(ftp)(http) |
TXT |
| GSM755209_tsb_rc.pileup.txt.gz |
565.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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