GSM5217238: TMZ group, 2_01; mouse gut metagenome; OTHER
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.