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| Status |
Public on Apr 01, 2019 |
| Title |
Setlow_2018_08_27_S1 |
| Sample type |
SRA |
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| Source name |
Plates_7d_hp
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| Organism |
Bacillus subtilis |
| Characteristics |
strain: PS533
tissue: dormant spores prepared on plates
sample type: highly purified over 7 days
tissue type: intact spores
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| Treatment protocol |
For chemical decoating, highly purified spore preparations were treated for 30 min at 70°C in 0.1 M DTT, 0.1 M NaCl, 0.1 M NaOH, 0.5% SDS, washed 6x at 4°C with 0.15 M NaCl and then with water.
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| Growth protocol |
Spores were routinely sporulated at 37°C on 2xSG medium agar plates for 24 hrs or ~ 2 d, and harvested. Sporulation plates were also incubated for 24 h or ~2 days, spores harvested. Spores were purified over ~ 7 days by daily multiple washes with 4°C water and intermittent sonication for 1 min, obvious contaminating material washed off surfaces of spore pellets with a gentle stream of water, followed by centrifugation through 50% Histodenz (highly purified) or only washed 3 times by centrifugation with 4°C water with sonication in between centrifugation ("less well purified"). Spores also were prepared at 37°C in liquid 2xSG medium for 2 d, which is the minimum time to get release from the sporangium of > 90% of the spores from sporulating cells and then purified as descibed above for spores from plates.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted and purified from spores using Ambion RiboPure-Bacteria Kit (product # AM1925). RNA quality was analyzed on the Agilent TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA) using the RNA High Sensitivity assay. Only samples with Ribosomal Integrity Numbers (RINe) values above 7.0 were considered for library preparation
RNA libraries were prepared for sequencing using Illumina TruSeq Stranded mRNA Sample Preparation kit. Libraries were validated for length and adapter dimer removal using the Agilent TapeStation 4200 D1000 High Sensitivity assay, and then quantified and normalized using the dsDNA High Sensitivity Assay for Qubit 3.0 (Life Technologies, Carlsbad, CA, USA).
Illumina MiSeq v3 chemistry (paired end 2 x 75bp read length). Sequencing read depth was targeted at 10-15 million total paired end reads/sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
Setlow_2018_08_27_S1
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| Data processing |
Analysis of raw data was performed using the Assembler module in MacVector (v16.0.9) (MacVector, Inc., Apex, NC) using default parameters.
Raw paired-end read data from each library were aligned to the B. subtilis strain 168 reference genome (accession number AL009126.3) using Bowtie.
Depth of coverage, read count, Reads Per Kilobase of transcript per Million mapped reads (RPKM), and Transcripts Per Kilobase Million (TPM) values for each library were exported as text for comparison.
Mapped reads were visualized over the reference genome using the Map option in MacVector to obtain the coverage of reads over individual genes or putative operons.
Genome_build: Bacillus subtilis strain 168 reference genome (NCBI GenBank accession number AL009126.3)
Supplementary_files_format_and_content: abundance measurements
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| Submission date |
Jan 23, 2019 |
| Last update date |
Apr 02, 2019 |
| Contact name |
Peter Setlow |
| E-mail(s) |
[email protected]
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| Organization name |
UConn Health
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| Department |
MBB
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| Street address |
263 Farmington Ave.
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| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06030 |
| Country |
USA |
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| Platform ID |
GPL21373 |
| Series (1) |
| GSE125530 |
Analysis of the messenger RNAs in Spores of Bacillus subtilis |
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| Relations |
| BioSample |
SAMN10794046 |
| SRA |
SRX5283813 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3576617_Setlow_2018_08_27_S1_Coverage.txt.gz |
185.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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