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Sample GSM7164736

Query DataSets for GSM7164736

Status

Public on Aug 15, 2023

Title

30C, biol rep 1

Sample type

SRA

Source name

Bacteria

Organisms

Lentilactobacillus kefiri; Lactobacillus kefiranofaciens

Characteristics

cell type: Bacteria
strain: JCM5818 and JCM6985
genotype: WT
treatment: 30C

Treatment protocol

All samples were initially inoculated with a mixed culture of L. kefiri and L. kefiranofaciens inoculum blended from 3-day cultures. 20 mL MRS medium was inoculated with 0.5 mL of the combined inoculum and incubated for 3 days, one set of 10 at 30°C, and another set of 10 at 37°C. For controls, L. kefiri and L. kefiranofaciens were inoculated in triplicate for each temperature. The tubes were removed from the incubator after 3 days, and 0.1 mL of each sample was placed in a 96 well plate for OD (600 nm) analysis. Samples were then centrifuged (3000×g, 4°C, 10 min), and the supernatant was removed and frozen for further analysis. The pellet was washed in fresh MRS (pH 5.5), divided in three aliquots, re-pelleted in microcentrifuge tubes, and frozen at -80°C for subsequent transcriptomic, proteomic, and metabolomic analysis.

Growth protocol

Both L. kefiri and L. kefiranofaciens were separately activated on MRS (Difco, BD Diagnostic, MD, USA) agar plates and incubated anaerobically for 3 days at 30°C. A single colony from each microorganism was picked and transferred into 15 mL Lactobacillus MRS broth and incubated anaerobically at 30°C for 3 days. The two strains of bacteria were subcultured twice until they reached the exponential growth phase.

Extracted molecule

total RNA

Extraction protocol

RNA was isolated using Omega Biotek E.Z.N.A.® Total RNA Kit II (Omega Biotek, Inc., Norcross, GA) according to the manufacturer’s protocol. Isolated RNA sample quality was assessed by High Sensitivity RNA Tapestation (Agilent Technologies Inc., California, USA) and quantified by Qubit 2.0 RNA HS assay (ThermoFisher, Massachusetts, USA). Ribosomal RNA depletion was performed with Ribo-Zero Plus rRNA Removal Kit (Illumina Inc., California, USA).
Samples were randomly primed and fragmented based on manufacturer’s recommendation. The first strand was synthesized with the Protoscript II Reverse Transcriptase with a longer extension period, approximately 40 minutes at 42°C. All remaining steps for library construction were performed according to the NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina® (New England BioLabs Inc., Massachusetts, USA). Final quantity of the libraries was assessed by Qubit 2.0 (ThermoFisher, Massachusetts, USA) and quality was assessed by TapeStation D1000 ScreenTape (Agilent Technologies Inc., California, USA). Illumina® 8-nt dual-indices were used. Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina® [NovaSeq S4] (Illumina, California, USA) with a read length configuration of 150 PE for 20M PE reads per sample (10M in each direction).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

11_30

Data processing

Raw data was initially processed using FastQC (v0.11.9) and Trimmomatic (v0.40) to identify and remove overrepresented artificial sequences such as adapters.
Cleaned reads were further analyzed with Trimmomatic to remove low quality bases. We used the “maxinfo” filtering in palindrome mode with the default parameters. Reads were subject to a quality threshold of 25 and if the retained length was less than 40 bp they were removed from further analysis.
The quality of sequencing data at the sample level before and after trimming and filtering was assessed using our in-house-developed method FQStat. All samples yielded high quality sequences and were carried on for downstream analysis.
Transcript quantification was done using Salmon (v1.6.0) (based on EnsemblBacteria assemblies for the two species. Transcripts per million (TPM) was used as the normalized abundance value for the transcripts.
Assembly: gca_002276955 and gca_900103655
Supplementary files format and content: Tab-delimited text file includes TPM values for each sample

Submission date

Apr 12, 2023

Last update date

Aug 15, 2023

Contact name

Hasan Otu

E-mail(s)

hotu2@unl.edu

Organization name

University of Nebraska-Lincoln

Department

Electrical and Computer Engineering