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| Status |
Public on May 07, 2019 |
| Title |
A56720_GGCTAC [Fn7_1 control] |
| Sample type |
SRA |
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| Source name |
RNA extracted from bacteria grown in broth culture
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| Organism |
Fusobacterium nucleatum |
| Characteristics |
strain: 7_1
growth medium: tryptic soy broth
experimental condition: control
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| Treatment protocol |
Caco-2 cells were grown to 85% confluence, washed briefly with PBS and resuspended in supplemented DMEM without antibiotics. They were then infected to a MOI of 100:1 with Fn 7-1 that had been grown to late log phase in TSBsupp, and incubated at 37°C in 5% CO2 for 4 hours 14. Cells were then washed with sterile PBS and treated with fresh DMEM containing gentamicin (0.5mg/mL) (Sigma-Aldrich) for 30 minutes at 37°C in 5% CO2 to kill any bacteria present outside of the Caco-2 cells. The Caco-2 cells were then trypsinized and quenched. All subsequent steps were carried out using reagents at 4°C. Cells were briefly pelleted by centrifugation before lysis with 0.1% (v/v) Nonidet P-40 (Sigma-Aldrich), aided with gentle mixing to release internalized (invasive) Fusobacterium cells. Cells were once again pelleted by centrifugation and washed briefly with 1X Versene. RNA was stabilized through the addition of TRIzol® Reagent (1mL per 50-100mg of pelleted sample) (ThermoFisher), before storage at -80°C. RNAlater® (Qiagen) was used in all buffers throughout the extraction process. Total RNA extraction was performed within 15 minutes to ensure minimal transcriptional changes during the process.
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| Growth protocol |
Propagation of Fn strains was carried out on fastidious anaerobic agar (FAA) plates (Neogen) supplemented with 5% [v/v] defibrinated sheep blood (ThermoFisher), or in tryptic soy broth supplemented with hemin (5µg/mL) and menadione (1µg/mL) (TSBsupp) (Sigma-Aldrich), with incubation in a humidified anaerobic chamber (Ruskinn Bug Box) at 37°C under an atmosphere of N2:CO2:H2 90:5:5. For infection assays, Fn 7-1 and Fn7-3 were grown in TSBsupp to late log phase and normalized for cell number using McFarland standards. Caco-2 human colon adenocarcinoma cells (American Type Culture Collection (ATCC), Manassas, VA line HTB37™) were used for in vitro infection assays. Cells were cultured in Dulbecco’s Modified Eagle Media (DMEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher), 10mM sodium pyruvate (Sigma-Aldrich), and 5µg/mL Plasmocin (Invitrogen) at 37°C in 5% CO2. For consistency, and to avoid genotypic and phenotypic variation caused by genetic drift, only Caco-2 cells from passages 4-12 were used for experiments.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was also isolated from TRIzol® -stabilized samples of homogenized bacterial/tissue culture cells from the following control experiments: 1) Fn 7-1 grown in TSBsupp; 2) Caco-2 cells, with no bacterial exposure; and 3) Caco-2 cells infected with Fn 7-1 cells for 4 hours, collected after trypsinization but without gentamicin treatment. The parameters of control #3 will resolve for Fn 7-1 genes that are needed for bacterial invasion into the host cell. The protocol for RNA isolation of all invasion and adhesion samples, and the respective controls were repeated with three biological replicates.
Samples were thawed, homogenized, and RNA was separated from DNA and cellular debris by adding 0.2 mL of chloroform per 1 mL of TRIzol® Reagent followed by vigorous vortexing. Samples were then centrifuged at 12,000 x g for 15 minutes at 4°C, the aqueous phase was recovered, and RNA was precipitated by adding 100% isopropanol followed by centrifugation at 12,000 x g for 10 minutes at 4°C. The RNA pellet was washed three times in 75% EtOH, air dried for 5 minutes and re-suspended in 50 µL nuclease-free, de-ionized H2O. Samples were treated with the Turbo DNA-free™ kit (ThermoFisher), the bacterial RNA fraction was enriched using the MICROBenrichTM kit, and the sample was then ribodepleted with the MICROBexpress™ kit (ThermoFisher), all following the manufacturer’s protocols. Finally, the quantity and quality of each RNA sample was assessed Agilent Bioanalyzer 2100 RNA Nanochips.
cDNA libraries (including biological replicates) containing both human and fusobacterial RNA were generated and sent for sequencing. The sequencing depth required for the accurate representation of both the bacterial pathogen and the mammalian host was set at a threshold of 200 million reads using estimates from previous studies. Library construction and Illumina sequencing were performed as previously described but with the following modifications: (1) each paired-end library was PCR-amplified for 15 cycles using the standard Illumina PE1 PCR primer; and (2) a modified PE2 primer including a unique six base insertion as an index sequence was used. Libraries were gel-purified to remove residual adapter dimers and then sequenced on the Illumina HiSeq™ 2000 platform. Four paired-end 100 nt sequence lanes were run per library for a total of 12 lanes, yielding 952.6 million raw read pairs, well above the 200 million read threshold.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
C8ACNANXX_7_GGCTAC
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| Data processing |
BWA alignment version 0.5.7 was used to generate global alignments on the paired-end reads24. BWA was run with the default parameter settings used for alignments, except that the "-s" option (to disable Smith-Waterman alignment) was used because this feature was not designed to handle the insert size distribution that occurs in paired-end RNA-Seq data. In the final stage of the initial alignment, all reads were excluded that failed Illumina's Chastity filter and turned on bit 512 in that record's bitwise flag to indicate the read failed platform/vendor quality checks. For stranded gene coverage analysis, the annotations came from NCBI (GCF_000158275.2_ASM15827v2_genomic.Fna) and correspond to the annotations in v30 of bacteria.ensembl.org. These annotations all include one annotation per gene and in this case NORM_TOTAL included all reads mapped to exons (since there was no mito sequence used for alignment). To determine each gene’s raw read count (each exons total unique reads) the RPKM strategy was used with the following formula25: (number of reads mapped to all exons in a gene x 1,000,000,000)/(NORM_TOTAL x sum of the lengths of all exons in the gene ) where NORM_TOTAL = the total number of reads that are mapped to exons (i.e. fractional read count for exons).
Genome_build: The Illumina paired-end sequence data were analyzed with BWA alignment software to map each read pair onto the GCF_000158275.2_ASM15827v2 genomic reference fasta for the subspecies Fn 7-1, which was downloaded from the NCBI genome browser (https://www.ncbi.nlm.nih.gov) in April 2016.
Supplementary_files_format_and_content: RPKM
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| Submission date |
May 04, 2019 |
| Last update date |
May 07, 2019 |
| Contact name |
Kyla Cochrane |
| E-mail(s) |
[email protected]
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| Organization name |
NuBiyota
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| Department |
Bioinformatics
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| Lab |
Dr. Emma Allen-Vercoe
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| Street address |
50 Stone Road East
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| City |
Guelph |
| State/province |
Ontario |
| ZIP/Postal code |
N1G 2W1 |
| Country |
Canada |
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| Platform ID |
GPL26625 |
| Series (1) |
| GSE130714 |
A survey of Fusobacterium nuceatum genes modulated by host cell infection |
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| Relations |
| BioSample |
SAMN11571757 |
| SRA |
SRX5788190 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3752363_A56720_1_lanes.stranded_collapsed_coverage.transcript.normalized.txt.gz |
91.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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