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Sample GSM8348567

Query DataSets for GSM8348567

Status

Public on Jun 26, 2024

Title

Lg-1POAb

Sample type

SRA

Source name

CM2267_MRS2

Organism

Lactobacillus gasseri

Characteristics

cell line: CM2267_MRS2
cell type: bacterial isolate
genotype: WT
treatment: 3.2 mM Palmitoleic acid
time: 1 hour

Treatment protocol

Representative strains were grown to exponential phase in MRS broth supplemented with L-cysteine (4 mM) and L. glutamine (1.1 mM), then treated with OA (3.2 mM), LOA (3.2 mM), POA (3.2 mM), or no treatment for 1 hour. Three culture replicates were performed for each condition. After treatment, cultures were pelleted and resuspend in trizol for bulk RNA-sequencing. Bulk RNA-sequencing and DESeq246 analysis identified genes differentially expressed (DE) in each species for each fatty acid treatment compared to no treatment controls. Strains were cultured anaerobically at 37C.

Growth protocol

All strain isolates All vaginal bacterial isolates were cultured under anaerobic conditions at 37–40°C using a palladium catalyst-based anaerobic chamber (COY) with an atmosphere of 5% carbon dioxide, 5% hydrogen, and 90% nitrogen (Airgas #X03NI90C3001054). Isolates were revived from frozen 25% glycerol stocks on MRS agar plates (Hardy Diagnostics #89407-144). For cultivation in liquid media, Lactobacillus MRS (MRS+CQ) broth supplemented with L-cysteine (4 mM) and L. glutamine (1.1 mM) in static culture was used. Starter cultures were grown for 48 hours.

Extracted molecule

total RNA

Extraction protocol

Trizol-preserved bacterial samples prepared as above were thawed, transferred to 2 mL FastPrep tubes (MP Biomedicals #115065002) containing 500 µL of 0.1 mm Zirconia/Silica beads (BioSpec Products #11079101z), and bead beaten for 90 seconds at 10 m/sec speed using the FastPrep-24 5G (MP Biomedicals #116005500) with a Metal QuickPrep adapter. After bead beating, samples were incubated on ice for 3 min, and 200 µL chloroform was added to each sample and mixed by tube inversion. After 3 min incubation at room temperature, samples were centrifuged (12,000xg, 15 min at 4°C) to form separated phase layers between the organic & aqueous sections. For each sample, 200 µL of the clear aqueous phase was transferred to separate clean tubes, mixed with an equal volume (200 µL) of 100% ethanol by tube inversion, and incubated for 5 min at RT. Using a Direct-zol RNA Purification kit (Zymo Research #R2070), each sample was then transferred to a Direct-zol spin column and centrifuged (16,500xg, 1 min at RT). Columns were washed twice with 400 µL Direct-zol RNA Pre-Wash Buffer, and once with 700 µL Direct-zol RNA Wash Buffer and then centrifuged at max speed for 2 min. To dry the pellet and remove any ethanol carryover, columns were centrifuged with lids open (12,000xg, 1 min at RT). Finally, columns were moved to RNase-free 1.5mL tubes, incubated with 100 µL of nuclease-free water for 5 min, and then eluted by centrifugation (12,000xg, 1 min at RT). Extracted RNA samples were kept on ice for use and QC or stored at –80°C.
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
Illumina cDNA libraries were generated using a modified version of the RNAtag-seq protocol94. Briefly, 250 ng of total RNA was fragmented, depleted of genomic DNA, dephosphorylated, and ligated to DNA adapters carrying 5’-AN8-3’ barcodes of known sequence with a 5’ phosphate and a 3’ blocking group (IDT). Barcoded RNA molecules were pooled and depleted of rRNA using the Pan-Bacteria riboPOOL depletion kit (siTOOLs Biotech, Galen Laboratories #dp-K096). Pools of barcoded RNAs were converted to Illumina cDNA libraries in 2 main steps: (i) reverse transcription of the RNA using a primer designed to the constant region of the barcoded adaptor with addition of an adapter to the 3’ end of the cDNA by template switching using SMARTScribe Reverse Transcriptase (Takara ClonTech #639538) as described95; (ii) PCR amplification using primers whose 5’ ends target the constant regions of the 3’ or 5’ adaptors and whose 3’ ends contain the full Illumina P5 or P7 sequences. cDNA libraries were sequenced on the Illumina NovaSeq SP 100 platform to generate paired end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

DESeq2
Since samples were barcoded in library preparation and then pooled for sequencing, reads from each sample were demultiplexed based on their associated barcode sequence using custom scripts. Up to 1 mismatch in the barcode was allowed, provided it did not assign the read a separate barcode included in the sequencing pool. Barcode sequences were trimmed from the first read, as were terminal G’s from the second read that may have been added by SMARTScribe during template switching.
For each sample, reads were aligned to the sample species reference genome (GCF_022455535.1 / ASM2245553v1 for L. crispatus; GCF_022456925.1 / ASM2245692v1 for L. gasseri; GCF_022456915.1 / ASM2245691v1 for L. jensenii) using BWA96 and read counts were assigned to genes and other genomic features using custom scripts. Differential expression analysis was conducted using DESeq246. The Benjamini-Hochberg procedure was used to control the false discovery rate (FDR) with ɑ=0.05.
Assembly: GCF_022455535.1_ASM2245553v1_translated_cds.faa
Assembly: GCF_022456915.1_ASM2245691v1_translated_cds.faa
Assembly: GCF_022456925.1_ASM2245692v1_translated_cds.faa
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

Submission date

Jun 24, 2024

Last update date

Jun 26, 2024

Contact name

Douglas S Kwon

E-mail(s)

[email protected]

Organization name

Ragon Institute

Lab

Kwon