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Sample GSM4735694

Query DataSets for GSM4735694

Status

Public on Aug 01, 2021

Title

143

Sample type

SRA

Source name

cecal patch

Organism

mouse gut metagenome

Characteristics

genotype: wt.wt
tissue: Cecal patch
gender: Male
treatment: Water

Treatment protocol

Gut inflammation was induced using dextran sodium sulfate (DSS). Adult wild-type and A30P α-syn mice (3-months of age) were exposed to a chronic DSS protocol that began at DSS concentration of 2.5% and increased to 4% over 4 cycles (+0.5% increment per cycle). One cycle consisted of 5 days of DSS, followed by 2 days of water (DSS: 160110, MP Biomedicals, LLC). The non-DSS colitis groups were administered normal drinking water. Mice were then given a 4-week long recovery period during which they received normal drinking water, followed by tissue harvest. Mice were anesthetized with pentobarbital and transcardially perfused with PBS before tissue collection.

Growth protocol

A30P α-syn and wild-type mice have been maintained on a C57BL/6 background for over 10 generations.

Extracted molecule

genomic DNA

Extraction protocol

Cecal patch tissue was snap frozen and stored at -80°C until processing for DNA isolation. Frozen cecal patch samples were ground on liquid nitrogen into a fine powder. DNA was then isolated from the tissue powder using the Powersoil DNA isolation kit (Qiagen) according to manufacturer’s protocol and quality was determined by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).
The 16S rRNA concentration in each sample was confirmed by qPCR using the forward primer: 5’-TCCTACGGGAGGCAGCAGT, and reverse primer: 5’-GGACTACCAGGGTATCTAATCCTGTT. Cecal patch samples were then processed for sequencing according to the Illumina’s 16S Metagenomic sequencing library protocol (doc. #15044223). Briefly, 16S rRNA was amplified from samples using forward primer: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, and reverse primer: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. The 16S rRNA V3-V4 region amplicon (550 bp) was verified by gel electrophoresis. The amplicon was then purified using AMPureXP beads. The purified amplicon was indexed using Nextera XT index kit according to the manufacturer’s protocol. Sample libraries were purified with AMPure XP beads, verified with an Agilent 2100 Bioanalyzer, and quantified with a Qubit dsDNA HS kit on a Qubit 3.0 fluorometer (Thermo Fisher Scientific). Sample libraries were then pooled in equimolar amounts.
The Michigan State University Genomics Core performed pooled library quality and quantification using a combination of the Qubit dsDNA HS kit, Advanced Analytical Fragment Analyzer High Sensitivity DNA NGS and Kapa Illumina Library Quantification qPCR assays. Sequencing was performed on a Illumina MiSeq v3 flow cell, loading a library concentration of 3 pM and a 30% spike-in of the Illumina PhiX control DNA library. Sequencing was performed in a 300 bp paired-end format on an Illumina MiSeq. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina MiSeq

Description

level-1.csv
level-2.csv
level-3.csv
level-4.csv
level-5.csv
level-6.csv
level-7.csv
feature-table.biom.tsv

Data processing

Library strategy: 16S rRNA sequencing
For microbiome data analysis, we first removed adapters and low quality reads (Q<30) from the sequencing reads with Trim Galore (v0.4.4). QIIME2 was used to profile the microbial community. In QIIME2, reads were first denoised and dereplicated with the dada2 algorithm prior to being classified against the SILVA database (v132) with a clustering at 99% sequence identity criterion.
For microbial pathway analysis, KEGG pathway abundances were estimated using the PICRUSt2 software.
Supplementary_files_format_and_content: The taxa abundance data for each level is in .csv format, with each row representing sample ID, each column represents taxa ID.
Supplementary_files_format_and_content: KEGG abundance data is in .tsv format, with each row represents KEGG ID, each column represents sample ID.

Submission date

Aug 21, 2020

Last update date

Aug 01, 2021

Contact name

Peipei Li

E-mail(s)

[email protected]

Phone

6162345547

Organization name

Van Andel Research Institute