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| Status |
Public on Jun 11, 2024 |
| Title |
S. enterica wild-type, 37°C, replicate A |
| Sample type |
SRA |
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| Source name |
14028s
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| Organism |
Salmonella enterica |
| Characteristics |
strain: 14028s
genotype: wild-type
treatment: 37°C
biological replicate: A
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| Treatment protocol |
At time 0, samples were taken and cultures shifted to 15°C. Cells grew at 15°C for 3 hours, and then sampleres were taken again
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| Growth protocol |
Bacteria were grown in LB at 37°C
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| Extracted molecule |
total RNA |
| Extraction protocol |
1.5 mL of bacterial culture was pelleted and resuspended in 500 μL of Trizol pre-heated to 65°C and then stored at -80°C until use. Samples were purified on a column using the Zymo Direct-Zol RNA Miniprep kit. They were then treated twice with DNase I using Turbo DNA-free (Invitrogen) for 30 minutes each at 37˚C to ensure that all DNA was degraded. The samples were further purified on a column using the Zymo RNA Clean and Concentrator Kit and again stored at -80˚C until sequencing.
Illumina cDNA libraries were generated using a modified version of the RNAtag-seq protocol. Briefly, 500 ng to 1 μg of total RNA was fragmented by heating to 94°C in NEB Alkaline Phosphatase Buffer, depleted of any residual genomic DNA, dephosphorylated, and ligated to DNA adapters carrying 5’-AN8-3’ barcodes of known sequence with a 5’ phosphate and a 3’ blocking group. Barcoded RNAs were pooled and depleted of rRNA using the RiboZero rRNA depletion kit (Epicentre). Pools of barcoded RNAs were converted to Illumina cDNA libraries in 2 main steps: (i) reverse transcription of the RNA using a primer designed to the constant region of the barcoded adaptor with addition of an adapter to the 3’ end of the cDNA by template switching using SMARTScribe (Clontech) as described (23); (ii) PCR amplification using primers whose 5’ ends target the constant regions of the 3’ or 5’ adaptors and whose 3’ ends contain the full Illumina P5 or P7 sequences. cDNA libraries were sequenced on the Illumina [NextSeq 500] platform at the Broad Institute to generate paired end reads.
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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 |
Sequencing reads from each sample in a pool were demultiplexed based on their associated barcode sequence using custom scripts. Up to 1 mismatch in the barcode was allowed provided it did not make assignment of the read to a different barcode possible. Barcode sequences were removed from the first read as were terminal G’s from the second read that may have been added by SMARTScribe during template switching.
Reads were aligned to the reference genomes using BWA v0.7.17 . Reads were counted with htseq-count v2.0.1
Assembly: 6 species are present here, so there are six reference sequences: GCF_000005845.2,GCF_000022165.1,GCF_000027085.1,GCF_000281535.2,GCF_000025565.1,GCF_000513215.1
Supplementary files format and content: Processed files are text files of (unnormalized) counts for each sample.
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| Submission date |
May 15, 2024 |
| Last update date |
Jun 11, 2024 |
| Contact name |
Daniel Stoebel |
| E-mail(s) |
stoebel@g.hmc.edu
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| Organization name |
Harvey Mudd College
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| Department |
Biology
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| Street address |
301 Platt Blvd
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| City |
Claremont |
| State/province |
CA |
| ZIP/Postal code |
91711 |
| Country |
USA |
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| Platform ID |
GPL27048 |
| Series (1) |
| GSE267531 |
The transcriptional response to low temperature is weakly conserved across Enterobacteriales |
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| Relations |
| BioSample |
SAMN41402824 |
| SRA |
SRX24559776 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8268275_A_JH03_0_counts.txt.gz |
18.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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