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| Status |
Public on May 20, 2021 |
| Title |
L. casei BL23_mDOT-seq U_replicate 1 |
| Sample type |
SRA |
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| Source name |
cells
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| Organism |
Lacticaseibacillus casei |
| Characteristics |
strain: BL23
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| Growth protocol |
Lactobacillus casei BL23 was grown in 200 ml of BD™ Difco™ Lactobacilli MRS Broth at 37 °C to an OD 600 nm of 0.2, 0.9, 3.1, 6.0, 8.5 and 10.0.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from bacteria collected at six growth points with RNAzol RT (RN 190) (Molecular Research Center, Inc.). Traces of DNA were removed with DNase I, RNase free (Thermo Scientific). rRNAs were depleted using the modified RiboMinus Transcriptome Isolation Kit, bacteria (Invitrogen). 50-500 nt RNAs were isolated after polyacrylamide gell electrophoresis and treated with RNA 5´ Pyrophosphohydrolase (RppH) (New England Biolabs).
For mDOT-seq U and C libraries RNA was denaturated in the presence of EDTA, modified with azide group and attached to 3' alkyne-adapter/RT primer via the click reaction. The resulting RNA-DNA conjugate was further processed according to the NEXTflex Small RNA-Seq Kit v3 (PerkinElmer) starting from the Step B with couple modifications listed: at Step D ¼ dilution of NEXTflex 5' 4N adapter was used, at Step E incubation at 42 °C was extended to 60 min, at Step G 15 PCR cycles were performed and Steps F and H1 were carried out as described in alternative protocol for Preparing Libraries without Size Selection. For N libraries, RNA was processed according to the NEXTflex Small RNA-Seq Kit v3 (PerkinElmer) with modifications in Steps E, F and H1 identical to mDOT-seq protocol except for Step G were only 12 PCR cycles were performed.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
BL23 U-1
50-500 nt RNA
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| Data processing |
Sequenced reads were trimmed for adaptors and low quality bases using cutadapt (v2.9) and deduplicated using BBMAP package (v38.41). UMI extraction was performed with UMI-tools (v1.0.1).
Reads were mapped to L. casei BL23 reference genome using hisat2 (2.2.0, options –no-spliced-alignment -a -I 0 -X 1000).
sRNAs were predicted using R (3.5.1) package APERO (1.0.3) and manually curated.
Raw counts were generated using featureCounts from Rsubread package. CPM’s were generated using edgeR package (R 3.5.1)
Genome_build: NC_010999.1
Supplementary_files_format_and_content: Tab-delimited text files include raw counts of all predicted sRNAs for all samples.
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| Submission date |
Feb 17, 2021 |
| Last update date |
May 20, 2021 |
| Contact name |
Kotryna Kvederaviciute |
| E-mail(s) |
kotryna.kvederaviciute@mif.vu.lt
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| Organization name |
Vilnius University
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| Department |
Life Sciences Center
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| Lab |
Institute Of Biotechnology
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| Street address |
Sauletekio av. 7
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| City |
Vilnius |
| ZIP/Postal code |
10257 |
| Country |
Lithuania |
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| Platform ID |
GPL30169 |
| Series (2) |
| GSE166930 |
High-throughput sequencing of Lactobacillus casei BL23 sRNAs |
| GSE166932 |
Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs in probiotic Lactobacillus casei |
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| Relations |
| BioSample |
SAMN16815316 |
| SRA |
SRX9518715 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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