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| Status |
Public on Dec 15, 2021 |
| Title |
Lactobacillus rhamnosus ATCC 53103-RNA-seq data-OS1 |
| Sample type |
SRA |
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| Source name |
yogurt
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| Organism |
Lacticaseibacillus rhamnosus GG |
| Characteristics |
cell type: whole cells
treatment: treated with osmotic stress (OS)
fraction: total mRNAs
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| Treatment protocol |
The growth of L. rhamnosus ATCC 53103 was monitored by automatic growth curve analyzer (Bioscreen Cpro; OY Growth Curves, Finland) at 600 nm (OD600). L. rhamnosus ATCC 53103 was growth in MRS broth at 37 ℃. The cells were harvested when the OD600 reached 1.8 by centrifugation (6000 × g, 10 min, 4 ℃). Then the cells were resuspended in two kinds of MRS broths which containing 0.0 and 0.6 M sodium lactate, respectively. For acid stress, the cells were collected when the OD600 reached 1.8 by centrifugation (6000 × g, 10 min, 4 ℃) and then resuspended in MRS medium (pH 5.0). The effect of acid stress on cell growth was further monitored. The effect of osmotic stress on cells growth was further monitored. Added 200 µg/ml chloramphenicol into the cultures when OD600 reached ~1.0, and then harvested cells after shaking for 2 min.
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| Growth protocol |
L. rhamnosus ATCC 53103 was obtained from the American Type Culture Collection (Manassas, VA). Cultures were stored at -80 ℃ in MRS medium containing 10% glycerol. The strain was revitalized in MRS broth at 37 ℃ for 12 h three times before use.
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| Extracted molecule |
total RNA |
| Extraction protocol |
L. rhamnosus ATCC 53103 lysate was prepared following the protocols described previously with minor modifications. Cultures were poured onto chipped ice of the same volume and centrifuged at 4000 × g for 10 min at 4 ℃ for harvest. The resulting cell pellet was washed using 10 ml resuspension buffer [10 mM MgCl2, 100 mM NH4Cl, 20 mM Tris-HCl (pH 8.0) and 1mM chloramphenicol], and centrifuged again at 3000 × g for 5 min at 4 ℃. Cell pellet of each sample was then resolved in 2 ml lysis buffer [20 Mm Tris-HCl (pH 8.0), 100 mM NH4Cl, 10 mM MgCl2, 0.4% Triton X-100, 0.1% NP-40, 100 U/ml of RNase-free DNase Ⅰ (Roche), 0.5 U/µl of SUPERase In RNase inhibitor (Ambion) and 1 mM chloramphenicol]. Resuspended cells were poured over liquid nitrogen in 50 ml tubes, which were prepared with holes on the cap punched by a syringe needle. Then the tubes were placed at -80 ℃ for 30 min for nitrogen evaporation. Frozen cell pellets were pulverized using Freezer/Mill 6770 with the speed of impactor at 10 cycles per second for 2 min in two sets, transferred to a 50 ml tube, and stored at -80 ℃. After nitrogen evaporation, pulverized cells in 50-ml tubes were placed in water bath at 30 ℃ for immediate thaw, after which the cells were kept on ice for 10 min, and then transferred into a 1.5 ml Eppendorf tube. Lysate was aliquoted into 200 µl fractions, and flash frozen by liquid nitrogen and stored at -80 ℃ prior to further use.
50 µl or the cell lysate per sample was used to isolate total mRNAs with Gene JETRNA purification kit (Thermo Fisher Scientific, K0731). The rRNA removal for 5 µg total mRNAs was accomplished using the Ribo-ZeroTM MagneticKit for Gram-negative bacteria (Epicentre, MRZGN126). Afterward, RNAs were purified with RNA Clean & Concentrator kit (Zymo Research, R1015).RPFs from 100 µl lysate per sample were extracted as previously described. Notably, the concentration of Mg2+ in the lysate was supplemented to 15 mM to decrease the dissociation of 70S ribosomes. Clarified lysate was digested using 60 U MNase per one A260 unit of RNA with 5 mM CaCl2 in the reaction system, rotated at 1400 × g for 1 h at 25 ℃ in a thermomixer. The digestion was quenched with 6 mM EGTA. RPFs were isolated with Sephacryl S400 (GE, MicroSpin S-400 HR, 27514001) and purified using Acid Phenol: Chloroform extraction method prior to storage at -80 ℃ for further use. cDNA libraries of total mRNA and RPFs were constructed using the ARTseqTM Ribosome Profiling Kit (Epicentre, RPHMR12126) with different index tag at the reverse primers listed in Table X. Other primers used during the cDNA library construction were same as mentioned in the ARTseq Ribosome Profiling Kit (Epicentre RPHMR12126).
Prepared cDNA libraries were tested with Agilent 2100 bio analyzer for quality determination and then subjected to sequencing for quality determination and then subjected to sequencing with 50-bp single end using Illumina.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
all.genes.expression.txt
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| Data processing |
Prepared cDNA libraries were tested with Agilent 2100 bio analyzer for quality determination and then subjected to sequencing for quality determination and then subjected to sequencing with 50-bp single end using Illumina Hiseq 2000.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include count and FPKM values for each Sample
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| Submission date |
Nov 16, 2021 |
| Last update date |
Dec 15, 2021 |
| Contact name |
Zhen Feng |
| E-mail(s) |
[email protected]
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| Organization name |
Northeast Agricultural University
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| Street address |
Changjiang road 600
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| City |
Harbin |
| State/province |
Heilongjiang |
| ZIP/Postal code |
150030 |
| Country |
China |
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| Platform ID |
GPL30958 |
| Series (1) |
| GSE188929 |
Ribosome profiling and RNA sequencing reveal genome-wide cellular translation and transcription regulation under osmotic stress in Lactobacillus rhamnosus ATCC 53103 |
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| Relations |
| BioSample |
SAMN23176775 |
| SRA |
SRX13146982 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5692033_RNA-seq_data-OS1.txt.gz |
27.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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