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| Status |
Public on Aug 20, 2019 |
| Title |
LL195_a3 |
| Sample type |
SRA |
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| Source name |
culture in BHI plus acid
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| Organism |
Listeria monocytogenes |
| Characteristics |
strain: LL195
treatment: acid
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| Treatment protocol |
Acid stress was performed by adding anequal volume of BHI adjusted to pH 1.6 with HCl
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| Growth protocol |
growth in BHI at 37 degrees unitl early stationary phase
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| Extracted molecule |
total RNA |
| Extraction protocol |
After one hour of acid exposure, or the respective control condition in BHI without acid, all reactions were stopped by the addition of 1 ml of 10 % acid phenol-chlorophorm in ethanol (v/v) to the cultures, which were chilled to 4 ¡C immediately. To extract the total RNA, the cultures were pelleted at 20Õ000 x g at 4 ¡C, resuspended in 200 ml of 10 mM Tris-buffer (Sigma-Aldrich, Buchs, Switzerland) containing 20 mg/ml Lysozyme (Sigma-Aldrich, Buchs, Switzerland) and 50 ml of 20 mg/ml Proteinase K (Qiagen, Venlo, the Netherlands) and subsequently lysed for 30 min at 37 ¡C. Cell lysates were then mixed with 1 ml TRI-Reagent (Invitrogen by Thermo Fischer Scientific, Zug, Switzerland) and bead-beaten for 2 x 60 s with cooling on ice in between in beat-beating tubes containing 1.4 mm ceramic beads (MagNA lyser green tubes, Roche, Rotkreuz, Switzerland). Subsequently the samples were centrifuged at 16Õ000 x g at 4 ¡C and the supernatant was transferred to a new tube, where 500 ml of bromo-chloro-propane (BCP, ACROS Organics, Geel, Belgium) were added. After 10 min incubation at room temperature the tubes were centrifuged for 15 min at 16Õ000 x g at 4 ¡C and the top aqueous layer was transferred into a new tube. Nucleic acids were precipitated overnight in 2.5 ml isopropanol at -80 ¡C. The following day, nucleic acids were pelleted, washed with 5 ml 75 % ethanol, resuspended in nuclease free water and adjusted to 500 ng/ml using a nanodrop (Witec AG, Sursee, Switzerland). 3 ml Turbo DNAse (Invitrogen by Thermo Fischer Scientific, Zug, Switzerland) and its buffer were added to digest the genomic DNA for one hour at 37 ¡C, with the addition of another 3 ml Turbo DNAse after 30 min to ensure complete digestion of genomic DNA. The DNAse treatment was followed by a phenol-chloroform (Fluka, Bucharest, Romania) extraction and RNA precipitation in EtOH with 1.6% (v/v) 3M sodium acetate at -80 ¡C overnight. The RNA was pelleted and washed with 70 % ethanol, dried and resuspended in 100 ml nuclease free water. Total RNA quality was assessed by a nanodrop measurement and an Agilent 2100 Bioanalyzer pico Chip. Ribosomal RNA was depleted with Ribo-Zero rRNA Removal Reagents (Bacteria)-Low Input and the Magnetic Core Kit-Low Input (Illumina, San Diego, USA) according to the manufacturerÕs specifications, and purified using the Agencourt RNAClean XP Kit (Beckman Coulter Inc., Brea, CA). A pico chip run on the bioanalyzer 2100 confirmed the depletion of 16S and 23S rRNA.
Library preparation was done with an Illumina TruSeq RNA stranded kit according to the manufacturers instruction. All samples were pooled and sequenced on one lane of a Novaseq (paired-end, 150bp per read) at the Functional Genomics Centre ZŸrich.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Reads were aligned to their corresponding genomes using the BWA mem algorithm (version 0.7.17) with default settings. Samtools 1.9 was used to convert and sort the output and featureCounts 1.6.3 summarized the aligned reads. Normalization and statistical calculation of differentially expressed genes was done in R with the EdegR package
Genome_build: NC_019556.1 for LL195, NZ_QYGN00000000.1 for stain N12-0605 and NZ_QYDR00000000.1 for stain N13-1507
Supplementary_files_format_and_content: a .txt file containig following collumns: Geneid;Chromosone or contig;Start;End;Strand;Length;number of counts
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| Submission date |
Aug 19, 2019 |
| Last update date |
Aug 22, 2019 |
| Contact name |
Marc Stevens |
| E-mail(s) |
marc.stevens@uzh.ch
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| Organization name |
University of Zurich
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| Department |
Vetsuisse
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| Lab |
Institute for Food Safety and Hygiene
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| Street address |
Winterthurerstrasse 272
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| City |
Zürich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
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| Platform ID |
GPL27107 |
| Series (1) |
| GSE135966 |
Global transcriptional response of three highly acid-tolerant field strains of L. monocytogenes to HCl stress |
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| Relations |
| BioSample |
SAMN12600303 |
| SRA |
SRX6734467 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4038652_LL195_6_3a.txt.gz |
43.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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