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| Status |
Public on Sep 30, 2015 |
| Title |
Mycoplasma pneumoniae (strain 6009)_CRO6 |
| Sample type |
SRA |
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| Source name |
M. pneumoniae culture
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| Organism |
Mycoplasmoides pneumoniae |
| Characteristics |
strain: 6009
strain type: 2
country of isolation: France
year: 2011
isolated from: BAL (bronchoalveolar)
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| Treatment protocol |
No treatment was applied
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| Growth protocol |
M. pneumoniae was grown in 50 mL of modified Hayflick medium supplemented with glucose at 37ºC
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| Extracted molecule |
total RNA |
| Extraction protocol |
After growing M. pneumoniae for 6h at 37°C, cells were washed twice with PBS and lysed with 700 µl of Qiazol buffer. Then, samples were lysed with 700 µl of Qiazol buffer. RNA extractions were performed by using the miRNeasy mini Kit (Qiagen) following the instructions of the manufacturer.
Libraries for RNA-seq were prepared following directional RNA-seq library preparation and sequencing. Briefly, 1 µg of total RNA was fragmented to ~100-150 nt using NEB Next Magnesium RNA Fragmentation Module (ref. E6150S, NEB). Treatments with Antarctic phosphatase (ref. M0289S, NEB) and PNK (ref. M0201S, NEB) were performed in order to make the 5’ and 3’ ends of the RNA available for adapter ligation. Samples were further processed using the TruSeq small RNA Sample Prep Kit (ref. RS-200-0012, Illumina) according to the manufacturer's protocol. In summary, 3’ adapters and subsequently 5’ adapters were ligated to the RNA. cDNA was synthesized using reverse transcriptase (SuperScript II, ref. 18064-014, Invitrogen) and a specific primer (RNA RT Primer) complementary to the 3’ RNA adapter. cDNA was further amplified by PCR using indexed adapters supplied in the kit. Finally, size selection of the libraries was performed using 6% Novex® TBE Gels (ref. EC6265BOX, Life Technologies). Fragments with insert sizes of 100 to 130 bp were cut from the gel, and cDNA was precipitated and eluted in 10 µl of elution buffer. Double-stranded templates were cluster amplified and sequenced on an Illumina HiSeq 2000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
CRO6
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| Data processing |
Gene annotation was transferred from the reference (Mycoplasma Pneumoniae M129) on the indicated assembled strains by using blat / liftover.
Reads were aligned to the corresponding assembled strain with bowtie2 (version 2.2.5) and sorted with samtools (version 1.2)
"Bedtools coverage" (version 2.15.0) was used to calculate the coverage for each gene then used for calculating the logCPKM as indicated in the following formula
logCPKM=log2 ( coverage /( (tot coverage / 1,000,000) * gene size / 1,000)
Genome_build: Mycoplasma pneumoniae strains 6009 (LHQG00000000) and 2882 (LHPQ00000000) and annotations from NC_000912.1
Supplementary_files_format_and_content: tabular files reporting the gene name (column 1), the total gene coverage (column 2), and the logCPKM (column 3). In column 1, ncMPN is the identifier for non coding RNAs found in the transcriptome analysis of M. pneumoniae M129 strain.
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| Submission date |
Jul 28, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Luca Cozzuto |
| E-mail(s) |
luca.cozzuto@crg.eu
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| Organization name |
Centre for genomic regulation (CRG)
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| Department |
CRG core facilities
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| Lab |
Bioinformatics Unit
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| Street address |
Carrer Dr. Aiguader 88
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| City |
Barcelona |
| State/province |
Barcelona |
| ZIP/Postal code |
08005 |
| Country |
Spain |
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| Platform ID |
GPL20750 |
| Series (1) |
| GSE71467 |
Comparative “-omics” in Mycoplasma pneumoniae Clinical Isolates Reveals Key Virulence Factors |
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| Relations |
| BioSample |
SAMN03943238 |
| SRA |
SRX1122953 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1835181_CRO6_8357_TTAGGC_s_sn.bam_cpkm.txt.gz |
15.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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