 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 18, 2024 |
| Title |
OME 16 + FMT |
| Sample type |
SRA |
| |
|
| Source name |
murine feces
|
| Organism |
mouse gut metagenome |
| Characteristics |
mouse strain: C57BL/6J
age: 52-week old
Sex: male
treatment: FMT + EtOH
tissue: murine feces
run id: 147444e789756376e73f7185041bb65b4bfc58c5
barcode: barcode12
|
| Treatment protocol |
Fecal pellets were collected from 52 weeks-old female mice were pooled, mixed and homogenized in PBS at concentration at 1 g feces/10mL PBS (Day 0). Mixture was centrifuged at 500 rpm for 5 min at 4◦C and supernatants were collected and used for fecal microbiota transplantation (FMT). Fifty-two weeks-old male mice were treated with a high-dose of an antibiotics cocktail (ABx) before FMT (Day 1). ABx was prepared in sterile H2O at the following concentration: ampicillin (1 mg/ml), vancomycin (5 mg/ml), streptomycin (10 mg/ml), and metronidazole (10 mg/ml). This antibiotics cocktail combines b-lactam (ampicillin), glycopeptide (vancomycin), aminoglycoside (streptomycin) and nitroimidazole (metronidazole) antibiotics and was proved sufficient to decrease mouse gut microbial load within hours targeting against the full spectrum of bacteria including both gram positive (ampicillin and vancomycin) and gram negative (ampicillin and neomycin) strains. Each 52 weeks-old male mice received 150 μL of the supernatant by oral gavage once a day continuously for 3 days (Days 2–4). Acute intoxication with ethanol was performed 15 days after the FMT (Day 19) across all groups (female/OFE, male/OME and male+FMT/OME+FMT). Specifically, mice were fasted at night for 12 hours before they were fed in the morning with one oral dose of 30% EtOH (gavage 6 g/kg body weight) with a gavage needle (Kent Scientific, Torrington CT). Feces were collected 8 hours after EtOH challenge and submitted to microbiota analysis.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Murine fecal samples were collected for bacterial genomic DNA extraction using the centrifugal affinity column system in the PureLink Microbiome DNA Purificaction Kit (Invitrogen), according to the manufacturer's instructions. Quantity and purity of the extracted genomic DNA were evaluated with a Thermo Scientific NanoDrop and stored at -20ºC until used.
For long-read sequencing of 16S rRNA gene, 10ng of total genomic DNA were subjected to library construction using the 16S barcoding kit (SQK-16S024) according to Oxford Nanopore Technologies (ONT) protocol. Specifically, bacterial DNA was amplified using barcoded universal 16S primers (27F and 1492R) supplied by ONT with 5’-tags that facilitate the ligase-free attachment of rapid sequencing adapters required during nanopore library preparation. The barcoded libraries were loaded and sequenced on MinION flow cells (FLO-MIN106D R9.4.1; ONT) using the MinION-Mk1C instrument (ONT)
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
MinION |
| |
|
| Data processing |
After 24 h, a total of 398,221 nanopore reads in two pooled runs were obtained for the 11 individual samples of this study, with a median long-read length of 1.59 kb, consistent with the expected 16S rRNA size. Data acquisition, real-time analysis, base-calling, and data transmission of FASTQ files was operated using the MinKNOW (v22.10.7) operating software on the MinION-Mk1C sequencing device. The cloud-based EPI2ME Fastq 16S (v2023.04.21) algorithm was used for analytical workflow of 16S rRNA FASTQ files, including alignment and classification of reads into operational taxonomic units (OUT) to the species level, according to the NCBI 16S rRNA reference sequence database (containing over 26,000 curated type strain sequences of 16S rRNA from bacteria and archaea).
Assembly: 16S RefSeq
Supplementary files format and content: EPI2ME output files 16S: classification_16s_barcode.csv
Library strategy: long-read sequencing of 16S rRNA gene
|
| |
|
| Submission date |
Apr 08, 2024 |
| Last update date |
Apr 18, 2024 |
| Contact name |
SERGIO ROA |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Navarra
|
| Department |
Biochemistry and Genetics
|
| Lab |
Immunogenetics lab
|
| Street address |
Irunlarrea, 1
|
| City |
Pamplona |
| ZIP/Postal code |
31008 |
| Country |
Spain |
| |
|
| Platform ID |
GPL34367 |
| Series (1) |
| GSE263477 |
Fecal microbiota transplantation from female donors restores gut permeability and reduces liver injury and inflammation in middle-aged male mice exposed to alcohol |
|
| Relations |
| BioSample |
SAMN40874753 |
| SRA |
SRX24189439 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
