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| Status |
Public on Mar 17, 2019 |
| Title |
Smeg_hypoxia 15 h_polyphosphatase Rep2 |
| Sample type |
SRA |
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| Source name |
Bacteria
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| Organism |
Mycolicibacterium smegmatis |
| Characteristics |
strain: mc2155
growth phase: Hypoxia 15 hours after sealing Wayne model vials
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| Treatment protocol |
For hypoxia experiments, a protocol similar to the Wayne model (Wayne and Hayes, 1996) was implemented. Briefly, 60 ml serum bottles (Wheaton, product number 223746, actual volume to top of rim 73 ml) were inoculated with 36.5 ml of M. smegmatis culture with an initial OD = 0.01. The bottles were sealed with rubber caps (Wheaton, W224100-181 Stopper, 20 mm) and aluminum caps (Wheaton, 20 mm aluminum seal) and cultures were grown at 37◦C and 125 rpm to generate hypoxic conditions. Samples were taken at an early stage of oxygen depletion when growth had slowed but not completely stopped (15 h) and at a later stage when a methylene blue indicator dye was fully decolorized and growth had ceased (24 h).
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| Growth protocol |
Bacteria were grown in Middlebrook 7H9 supplemented with ADC (Albumin Dextrose Catalase, final concentrations 5 g/L bovine serum albumin fraction V, 2 g/L dextrose, 0.85 g/L sodium chloride, and 3 mg/L catalase), 0.2% glycerol and 0.05% Tween 80. For the exponential phase experiment (Dataset 1), 50 ml conical tubes containing 5 ml of 7H9 were inoculated with M. smegmatis to have an initial OD = 0.01. Cultures were grown at 37◦C and 200 rpm.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNAseq 5' end mapping library. 5' triphosphates were converted to 5' monophosphates by 5' Polyphosphatase before adapters were ligated to RNA 5' ends.
RNA was extracted as follows: frozen cultures stored at −80◦C were thawed on ice and centrifuged at 4,000 rpm for 5 min at 4◦C. The pellets were resuspended in 1 ml Trizol Life Technologies) and placed in tubes containing Lysing Matrix B (MP Bio). Cells were lysed by bead-beating twice for 40 s at 9 m/sec in a FastPrep 5G instrument (MP Bio). 300 µl chloroform was added and samples were centrifuged for 15 min at 4,000 rpm at 4◦C. The aqueous phase was collected and RNA was purified using Direct-Zol RNA miniprep kit (Zymo) according to the manufacturer’s instructions. Samples were then treated with DNase Turbo (Ambion) for 1 h and purified with an RNA Clean & Concentrator-25 kit (Zymo) according to the manufacturer’s instructions
RNA samples from each biological replicate were split in three, in order to generate two 50 -end differentially treated libraries and one RNAseq expression library. RNA for library 1 (“converted” library) was treated either with RNA 50 pyrophosphohydrolase RPPH (NEB) (exponential phase experiment), or with 50 polyphosphatase (Epicenter) (hypoxia experiment and normoxia controls), in order to remove the native 50 triphosphates of primary transcripts, whereas RNA for Library 2 (“nonconverted” library) was subject to mock treatment. Thus, the converted libraries capture both 50 triphosphates (converted to monophosphates) and native 50 monophosphate transcripts, while non-converted libraries capture only native 50 monophosphates. Library construction was performed as described by Shell et al. (2015). For RNA expression libraries, KAPA stranded RNA-Seq library preparation kit and NEBNext Ultra RNA library prep kit for Illumina (NEB) were used for Dataset 1 and Dataset 2, respectively, according to manufacturer’s instructions. The following major odifications were introduced into the protocols: (i) For RNA fragmentation, in order to obtain fragments around 300 nt, RNA was mixed with the corresponding buffer and placed at 85◦C for 6 min (Dataset 1), or at 94◦C for 12 min (Dataset 2). (ii) For library amplification, 10 or 19–23 PCR cycles were used for Dataset 1 and Dataset 2, respectively. The number of cycles was chosen according to the amount of cDNA obtained for each sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
wildtype Smegmatis mc2155. Hypoxia 15 hours 5' end mapping library, treated with polyphosphatase, replicate 2
Middlebrook 7H9 supplemented with ADC (Albumin Dextrose Catalase, final concentrations 5 g/L bovine serum albumin fraction V, 2 g/L dextrose, 0.85 g/L sodium chloride, and 3 mg/L catalase), 0.2% glycerol and 0.05% Tween 80
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| Data processing |
bwa and samtools were used to generate .bam and .sam files from fastq files
Read pairs that were not correctly oriented with respect to each other were filtered out, unpaired reads were filtered out, and the region encoding the rRNA was filtered out
For expression libraries, coverage at each genome coordinate and RPKMs were calculated.
For 5' end libraries, the coverage of each position at the M. smegmatis genome was calculated.
Genome_build: NC_008596
Supplementary_files_format_and_content: RPKM files contain RPKM data from RNAseq expression libraries
Supplementary_files_format_and_content: DEseq excel file contains output from DEseq2 differential expression analysis (normoxia vs hypoxia)
Supplementary_files_format_and_content: Coverage files for RNAseq libraries contain coverage at every nt acrosss the genome (not normalized)
Supplementary_files_format_and_content: 5-prime-end coverage files contain read depth at each RNA 5' end identified in 5' end mapping libraries (not normalized)
Supplementary_files_format_and_content: 5-prime-end excel file contains normalized coverage at 5' ends that met filtering criteria as detailed in Martini et al 2019 Frontiers in Microbiology
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| Submission date |
Mar 16, 2019 |
| Last update date |
Mar 18, 2019 |
| Contact name |
Scarlet Shell |
| E-mail(s) |
[email protected]
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| Organization name |
Worcester Polytechnic Institute
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| Department |
Biology and Biotechnology
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| Lab |
Scarlet Shell
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| Street address |
60 Prescott St
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| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platform ID |
GPL25523 |
| Series (1) |
| GSE128412 |
Defining the transcriptional and post-transcriptional landscapes of Mycobacterium smegmatis in aerobic growth and hypoxia |
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| Relations |
| BioSample |
SAMN11155142 |
| SRA |
SRX5531125 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3673862_hyp_15_3_con_sb_minus.coverage.txt.gz |
16.7 Mb |
(ftp)(http) |
TXT |
| GSM3673862_hyp_15_3_con_sb_plus.coverage.txt.gz |
16.7 Mb |
(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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