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| Status |
Public on Feb 11, 2011 |
| Title |
t7hrs |
| Sample type |
SRA |
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| Source name |
Whole cell
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| Organism |
Legionella pneumophila |
| Characteristics |
strain: Phil-1.pMip-GFP
infection: none, BYE broth
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| Treatment protocol |
ribosomal RNA depleted total RNA
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| Growth protocol |
Legionella pneumophila Phil-1 WT and the constitutively GFP-expressing bacterial strain Legionella pneumophila Phil-1 pMip.gfp ( both kindly provided by Antje Flieger, Wernigerode, Germany) were cultured on buffered charcoal-yeast extract agar (BCYE, Sigma-Aldrich, Gillingham, UK) for 3 days at 37°C (15) or grown in buffered yeast extract (BYE) broth at 37°C with shaking at 250 rpm. Bacterial growth was monitored at an optical density of 600 nm (OD600) after inoculation to an OD600 of 0.1. Acanthamoeba castellanii Neff (ATCC 30010) was cultured in PYG medium (Formedium, Norfolk, UK) at 30°C. The infection assays were performed according to Moffat and Tompkins. In brief A. castellanii cells were washed in A. c. buffer and adjusted to 106 cells per mL. 10 mL amoebal suspension was transferred to a 75 cm2 tissue culture flask and incubated at 37°C for 1 h. Stationary phase L. pneumophila Phil-1 grown on BCYE agar, diluted in A.c. buffer, were mixed with A. castellanii at a MOI of 100, defining the start point of the time-course experiment. Subsequent to invasion for 1 h at 37°C A. castellanii cells were washed three times to remove external bacteria. The infection was monitored by fluorescence microscopy and viable cell counts of L. pneumophila on BCYE agar.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Strand specific RNAseq libraries were prepared for the Illumina GAIIx (Illumina Inc, San Diego, Ca, USA) using a variation on a previously published protocol (Croucher et al., Nucleic Acids Res 2009, 37:e148). Briefly the rRNA depleted RNA from each time-point was fragmented using divalent cations. First strand cDNA synthesis was carried out in a reaction containing Super Script II reverse transcriptase, random hexamer primers and dNTPs. First strand reaction components were removed with Illustra MicroSpin G-50 columns (GE Healthcare Biosciences, Pittsburg, PA, USA). dUTP containing second strand cDNA was generated with DNA Polymerase and RNaseH in a reaction mix containing dATP, dCTP, dGTP and dUTP but no dTTP. Products were further processed, end repaired, addition of a single A to the 3´-end and ligation of indexed adapters. 6 nt barcoded Illumina compatible adapters were utilized in place of the commercially supplied adapters (Craig et al., Nat Methods 2008, 5:887-893 2008). Libraries were sized selected (cut at 200+50 nt) on 2.5% TAE agarose gels. Finally prior to library amplification the dUTP containing second strand was removed via digestion with Uracil DNA Glycoylase (1 unit) (Bioline, London, UK). Clustering and sequencing of the material was carried out as per manufacturers instructions – v2 Single Read Cluster Kits and v3 SBS kits (Illumina Inc, San Diego, Ca, USA) were utilized for all 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 |
Reads were mapped to the L. pneumophila Phil-1 genome (NC_002942.5) using the Bowtie short read aligner (PMID: 19261174) with the “--best” flag
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| Submission date |
Feb 11, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
James Prendergast |
| Organization name |
MRC Human Genetics Unit
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| Street address |
Crewe Road
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| City |
Edinburgh |
| ZIP/Postal code |
EH42XU |
| Country |
United Kingdom |
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| Platform ID |
GPL13156 |
| Series (1) |
| GSE27232 |
Strand specific sequencing illustrates the complex transcriptional response of Legionella pneumophila during infection |
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| Relations |
| SRA |
SRX041877 |
| BioSample |
SAMN00210879 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM673302_t7_SORTED.bed.gz |
129.6 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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