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Sample GSM4634552

Query DataSets for GSM4634552

Status

Public on Sep 15, 2020

Title

H2O Control 1

Sample type

SRA

Source name

16S sequencing control with water only

Organism

blank sample

Characteristics

molecule subtype: 16S Amplicon
sample type: 16S sequencing control with water only

Treatment protocol

Each group recieved different treatments: naïve mice recieved no treatment, EAE (MOG+CFA) mice were immunized subcutaneously with 50 μg of myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) (CSBIO #CS0681) emulsified at a 1:1 ratio in Complete Freud’s Adjuvant (Sigma Aldrich #F5881). On days 0 and 2, 250 ng of pertussis toxin (List Biologicals #180) was administered intraperitoneally. CFA imce recieved only the equivalent dose of Complete Freud’s Adjuvant.

Growth protocol

Female C57BL/6 mice were maintained on a 12 hours light/dark cycle with lights on at 7am and provided with standard food and water ad libitum. Mice were housed at 2-3 per cage and experimental treatment was assigned randomly to each cage.

Extracted molecule

total RNA

Extraction protocol

Fecal samples were collected from live mice restrained with a tail scruff. DNA for 16S amplification was extracted with a phenol-chloroform method: Fecal pellets were placed in 2 mL tubes containing 200 μL silica-zirconia beads (0.1 mm). The tubes were 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.
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 (Forward Primer: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, Reverse Primer: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC). 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.
V3-V4 16S-seq

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina MiSeq

Description

H2O-1_S1_L001

Data processing

Demultiplexed FASTQ files were processed with the Dada2 v1.12.1 function "filterAndTrim": truncLen = c(300,250), maxN = 0, maxEE = c(10,10), truncQ = 2, rm.phix = TRUE, compress = TRUE, multithread = TRUE
Error learning with Dada2 v1.12.1 "learnErrors" for R1 and R2 reads: multithread = TRUE, Dereplication with "derepFastq": verbose = TRUE
Amplicon Sequence Variant Identification with Dada2 v1.12.1 "dada" from learned error rates and dereplicated FASTQ reads: multithread = TRUE
Read pair merging with Dada2 v1.12.1 "mergePairs": verbose = TRUE
Chimera identification and removal with Dada2 v1.12.1 "removeBimeraDenovo": nethod = "consensus", multithread = TRUE, verbose = TRUE
Taxonomy assignment with Dada2 v1.12.1 "assignTaxonomy": multithread = TRUE
Genome_build: Taxonomy was assigned manually with input from the following 16S reference databases: Silva v132, Greengenes v13_8_set_97, GTDB bac-arc_ssu_r86, and RefSeq RDB16S v2_May2018
Supplementary_files_format_and_content: comma separated files (.csv) containing: 1. ASV abundances per sample, 2. Taxonomy of each ASV, and 3. Sample parameter data and Identification

Submission date

Jun 23, 2020

Last update date

Sep 15, 2020

Contact name

David Michael Johanson

E-mail(s)

dmj6ab@virginia.edu

Phone

3307327182

Organization name

UVa School of Medicine