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Sample GSM4661468

Query DataSets for GSM4661468

Status

Public on Jul 08, 2020

Title

Sample 2_WT.1R

Sample type

SRA

Source name

WT_EAE_1_fecal pellet

Organism

Mus musculus

Characteristics

strain background: C57BL/6
age: 8 weeks
genotype: WT
treatment: EAE
experiment day: 1
sample type: fecal pellet

Treatment protocol

Chronic stress was started after at least 1 week of acclimation. All behavioral interventions were performed between 4 pm and 7 pm and take-downs were performed between 11am and 2 pm. Animals were excluded from the experiments if they developed illnesses (e.g. dermatitis) that might affect the outcome of the results. Mice were subjected daily to an acute stressor (1–2 hours: restraint, loud white noise, crowded housing, strobe light) and an overnight stressor (12–24 hours: 45° cage tilting, repeated cage changes, wet bedding, dark deprivation) presented in a randomized fashion. For restraint stress, mice were placed in clean 50 mL conical tubes with pierced holes for ventilation for 1 hour. For crowded housing stress, mice were placed on top of the cage wire for 2 hours, after which their cage was changed. For wet bedding stress, 200 mL of water was added to the bedding of a clean cage. All procedures used autoclaved, sterile materials (bedding, water) in order to prevent contamination.

Growth protocol

The mice were maintained on a 12 hours light/dark cycle with lights on at 7am. Animals were housed 2–3 per cage and cages were randomly assigned to control or experimental groups.

Extracted molecule

genomic DNA

Extraction protocol

Whole genomic DNA was isolated via phenol-chloroform extraction. Briefly, a fecal pellet was placed in a 2 mL tube containing 200 μL silica-zirconia beads (0.1 mm). The tube was filled with 750 μL extraction buffer, 200 μL 20% SDS and 750 μL phenol-chloroform-isoamyl alcohol (25:24:1). After disruption, the aqueous phase was separated by centrifugation and cleaned up with two washes of chloroform-isoamyl alcohol (24:1). The DNA was precipitated and resuspended in 10 mM Tris solution.
For 16S rRNA sequencing, the V3-V4 region of the 16S rRNA gene was amplified for 25 cycles using specific primers with adapter overhangs as per the Illumina library preparation guide. Following purification of the PCR products, individual indexes were added to the amplicons by PCR. The amplicons were purified, pooled in equal quantities, and then sequenced on the Illumina MiSeq platform.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina MiSeq

Data processing

Reads with an average quality score below 25 (from any 10-bp window) or a mismatched barcode were removed
Paired-end reads were then merged using the software FLASH
Merged reads were analyzed using the QIIME pipeline with default parameters to remove chimeric, pick 97%-identity OTUs and assign taxonomy
Genome_build: Greengenes 13_8
Supplementary_files_format_and_content: csv: Includes OTUs and abundances that were detected in the dataset as well as taxonomic lineages for OTUs

Submission date

Jul 07, 2020

Last update date

Jul 09, 2020

Contact name

David Michael Johanson

E-mail(s)

dmj6ab@virginia.edu

Phone

3307327182

Organization name

UVa School of Medicine