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| Status |
Public on May 05, 2021 |
| Title |
B. subtilis PS533_Spores_b |
| Sample type |
SRA |
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| Source name |
B. subtilis PS533
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| Organism |
Bacillus subtilis |
| Characteristics |
strain: PS533
tissue: Spores
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| Treatment protocol |
Spores of all species were scraped from plates into 4 ˚C water and highly purified over ~7 d as described previously(6,7,8), including a final purification by centrifugation through a very high density (Histodenz) medium to remove spore associated debris as much as possible. Final spore preparations were >98% free from growing or sporulating cells and germinated spores as determined by phase contrast microscopy and spores pelleted by centrifugation had no obvious debris on the pellet surface. Spores were stored in water at 4 ˚C protected from light. 6. Crawshaw AD, Serrano M, Stanley WA, Henriques AO, Salgado PS. 2014. A mother cell-to-forespore channel: current understanding and future challenges. FEMS Microbiol Lett 358:129-136.
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| Growth protocol |
Spores of B. subtilis PS533 B. atrophaeus ATCC 9372 and B. thuringiensis Al Hakam were prepared as previously described on double strength Schaeffer's glucose agar plates(1,2) which were incubated for ~48 hours at 37 ˚C. 1. Nicholson WL, Setlow P. 1990. Sporulation, germination and outgrowth, p 391-450. In Harwood CR, Cutting SM (ed.), Molecular biological methods for Bacillus. John Wiley and Sons, Chichester, UK. 2. Paidhungat M, Setlow B, Driks A, Setlow P. 2000. Characterization of spores of Bacillus subtilis which lack dipicolinic acid. J Bacteriol 182:5505-5512.
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| Extracted molecule |
total RNA |
| Extraction protocol |
1 ml of each strain of dormant spores at an OD600 of 25 was pelleted by centrifugation. Prior to pelleting, to get a more accurate quantitation of the number of spores present, spore samples were diluted 1 to 25 in water, to give an OD600 of ~1.0, and the numbers of spores were counted in a Petroff-Hausser chamber to determine spores per ml. Spores were disrupted by shaking with glass beads in a Mini-Bead-beater (Biospec Products, Bartlesville, OK) for 1 min, followed by 1 min on ice with a total of two disruption periods, and giving >98% spore breakage (9). RNA was extracted using the RiboPure – Bacteria kit (Thermo Fisher Scientific, Waltham, MA) as previously described (9). Total RNA was quantitated using the Qubit RNA HS (High Sensitivity) Assay Kit (Thermo Fisher Scientific) (10) with the Qubit Fluorometer, using appropriate dilutions to get measurements that fell within the standard curve (500 ng/mL maximum concentration). Generally, a dilution range of 1:500 to 1:1000 of the purified initial total RNA gave quantifiable values. From this obtained concentration and the previously counted spore value, values of total RNA per spore were determined. 9. Korza G, Setlow B, Li Q, Rao L, Setlow P. 2016. Changes in Bacillus small molecules, rRNA, germination and outgrowth after extended sublethal exposure to various temperatures: evidence that protein synthesis is not essential for spore germination. J Bacteriol 198:3254-3264. 10. Garcia-Elias A, Alloza L, Puigdecanet E, Nonell L, Tajes M, Curado J, Enjuanes C, Diaz O, Bruguera J, Martí-Almor J, Comin-Colet J, Benito B. 2017. Defining quantification methods and optimizing protocols for microarray hybridization of circulating microRNAs. Sci Rep 7:7725.
Total RNA extracted and purified from spores of various species was submitted to University of Connecticut’s Center for Genome Innovation for ribosomal RNA depletion, RNA-seq library synthesis, and next-generation sequencing. Briefly, total RNA was ribo-depleted to remove most rRNA, the quality of the ribo-depleted RNA examined using the Agilent TapeStation 4200 D1000 high-sensitivity assay, and RNA-seq on RNA from all species was carried out on duplicate samples using an Illumina Next-Seq 500/550, all as described previously (11,12). Sequencing read depth was targeted at > 4 x 106 reads per sample. RNA-seq data were processed and analyzed as described previously (14) and levels of mRNA nt in an individual spore in populations were calculated using values for total RNA nt per individual spore determined as described above, and assuming that spore mRNA comprised 3% of total RNA (11). 11. Korza G, Camilleri E, Green J, Robinson J, Nagler K, Moeller R, Caimano MJ, Setlow P. 2019. Analysis of messenger RNAs in spores of Bacillus subtilis. J Bacteriol 201:e-00007-19. 12. Camilleri E, Korza G, Green J, Hui J, Li YQ, Caimano MJ, Setlow P. 2019. Properties of aged spores of Bacillus subtilis. J Bacteriol 201:e00231-19.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Files were concatenated.
Quality was assessed using FASTQC v011.8
Files trimmed Sickle single end V. 133
RPKM files were generated with EDGE_pro v1.3.1
Genome_build: ASM1506v1; ASM2582v1; ASM224349v1; ASM230013v1; ASM904v1; ASM208006v1
Supplementary_files_format_and_content: Coverage, #reads, RPKM
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| Submission date |
May 04, 2021 |
| Last update date |
May 05, 2021 |
| Contact name |
Peter Setlow |
| E-mail(s) |
setlow@uchc.edu
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| Organization name |
UConn Health
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| Department |
Molecular Biology and Biophysics
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| Lab |
Peter Setlow
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| Street address |
263 Farmington Ave
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| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06030-3305 |
| Country |
USA |
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| Platform ID |
GPL24109 |
| Series (1) |
| GSE173854 |
Levels and Characteristics of mRNAs in Dormant Spores of Firmicute Species |
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| Relations |
| BioSample |
SAMN19014914 |
| SRA |
SRX10773178 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5281859_sub_b_edge.out.rpkm_0.txt.gz |
53.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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