16S-seq of Mus musculus:Ctrl1.2
The total genomic DNA from the samples was extracted using the CTAB/SDS method. 16SV3-V4 regions were amplified using specific primers with barcodes. All the PCR reactions were carried out with Phusion High-Fidelity PCR Master Mix (New England Biolabs) and detected by agarose gel electrophoresis. The samples with a bright main strip between 400-450 bp were chosen for further experiments. The PCR products were mixed in equidensity ratios and purified with the Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturers recommendations, and the index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and the Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina HiSeq 2500 platform, and 250 bp paired-end reads were generated.