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Sample GSM5217238

Query DataSets for GSM5217238

Status

Public on Apr 28, 2021

Title

TMZ group, 2_01

Sample type

SRA

Source name

mouse feces

Organism

mouse gut metagenome

Characteristics

host: Specific-pathogen-free mouse
host age: 8-10 weeks
host strain: C57BL/6
host sex: male
group: TMZ group
treatment: TMZ gavage started at 7 days after implantation (T1) for five consecutive days (days 8-12 after implantation)
time point: T0 (the day of glioma cells implantation)
tissue: mouse feces

Treatment protocol

After a 7-day adaptive feeding, mice were randomly allocated to two groups (i.e., vehicle- and TMZ- treated groups, n=6/group) and underwent intracranial GL261 cells implantation. TMZ gavage started at 7 days after implantation (T1) with the dosage of 50 mg/kg/d for five consecutive days (days 8-12 after implantation). TMZ solution was freshly prepared for gavage. The vehicle-treated mice received the gavage of 0.5% sodium carboxymethyl cellulose. The stool samples from the mice were collected at four time points: days 1 (the day of glioma cells implantation, T0), 7 (the day before TMZ gavage, T1), 14 (7 days after first TMZ gavage, T2), and 28 (21 days after first TMA gavage, T3).

Extracted molecule

genomic DNA

Extraction protocol

Bacterial genomic DNA was extracted by the FastDNATM Spin Kit for Soil. The V3-V4 region of the bacterial 16S ribosomal DNA was amplified using the following primer pair: 341F and 805R.A synthetic spike-in plasmid was used as internal reference to quantitatively detect the total number of bacterial 16S rRNA genes.Sample (10 ng/μL) and spike-in internal reference DNAs were used as templates for PCR. 250x2 bp were sequenced using the Illumina MiSeq/HiSeq Benchtop Sequencer.
A total of 48 samples were collected, and bacterial genomic DNA was extracted by the FastDNATM Spin Kit for Soil (MP Biomedicals, USA) according to the manufacturer’s protocols. Extracted DNA was quantified using the Invitrogen Qubit3.0 Spectrophotometer (Thermo Fisher Scientific, USA). Quality and integrity of the DNA were evaluated using NanoDrop 2000 (Thermo Fisher Scientific, USA) followed by 1.5% agarose gel electrophoresis. Isolated DNA was stored at -20°C until processing. The V3-V4 region of the bacterial 16S ribosomal DNA was amplified using the following primer pair: 341F and 805R. A synthetic spike-in plasmid was used as internal reference to quantitatively detect the total number of bacterial 16S rRNA genes. PCR amplified products were purified using equal volume of Agencourt AMPure XP PCR Purification Beads (Beckman Coulter, USA). The mixed-sample library was identified using the Agilent 2100Bioanalyzer (Agilent Technologies, USA) to detect the size of the inserted fragments in the sequencing library (confirming no specific amplification between 120 and 200 bp) and the concentration of these sequencing inserts were quantified.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Data processing

The obtained sequencing reads were quality-filtered using the following steps: (i) the end bases with mass less than 20, possible adapter sequences, and sequences shorter than 100 bp were removed (TrimGalore v0.4.5); (ii) the paired sequences from double-terminal sequencing were spliced after which the merged low-quality sequences (base with mass less than 20 is more than 90%) were removed (FLASH2 v2.2.00); (iii) the primer sequences and sequences containing N bases/homopolymer >6 bp were removed (mothur v.1.39.3); (iv) the sequences with a total base error rate of >2 and length <100 bp were removed (usearch v10).
Operational taxonomic units (OTUs) were clustered using a similarity cut-off value of 97%, and chimeric sequences were removed after singleton removal (usearch v10).
A standard curve equation based on the spike-in sequence of each sample was created, and the absolute copy number of each OTU in each sample was calculated.
Sequences were aligned to the RDP 16S rRNA database (trainset 16/release 11.5) with a credibility of >80% using mothur v.1.39.3.
Genome_build: RDP 16S rRNA database
Supplementary_files_format_and_content: quantification_otu_table.txt (table contains OTU numbers in each sample with taxonomic annotation for absolute and relative quantification separately (spike-in sequence was not included)).

Submission date

Mar 29, 2021

Last update date

Apr 28, 2021

Contact name

Zhi-Qiang Li

E-mail(s)

[email protected]

Phone

0086 18907123005

Organization name

Zhongnan Hospital of Wuhan university