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Sample GSM5088624 Query DataSets for GSM5088624
Status Public on May 20, 2021
Title L. casei BL23_mDOT-seq C_replicate 2
Sample type SRA
Source name cells
Organism Lacticaseibacillus casei
Characteristics strain: BL23
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.
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.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description BL23 C-2
50-500 nt RNA
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.
Submission date Feb 17, 2021
Last update date May 20, 2021
Contact name Kotryna Kvederaviciute
Organization name Vilnius University
Department Life Sciences Center
Lab Institute Of Biotechnology
Street address Sauletekio av. 7
City Vilnius
ZIP/Postal code 10257
Country Lithuania
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
BioSample SAMN16815314
SRA SRX9518713

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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