GSM7461561: Young_CreER_F_2; Mus musculus; OTHER
Fresh fecal samples from 9- and 23-months old animals were obtained in the morning, immediately snap freeze in liquid nitrogen, and stored in -80℃. These samples were used for 16S rRNA gene analysis for microbiota profiling from the V1–V2 region of 16S rRNA genes. DNA extraction was performed using QIAamp DNA Stool Mini Kit (Qiagen) following the manufacture's instruction. DNA was eluted in 50 µl DNAse free water. Twenty nanograms of DNA were used for the amplification of the 16S rRNA gene with primers 27F-DegS (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTTYGATYMTGGCTCAG-3') and 338R I (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCWGCCTCCCGTAGGAGT-3') + 338R II (5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCWGCCACCCGTAGGTGT-3')87 for 25 cycles. Primers have Illumina sequencing index attached; Index forward (5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3') and index reverse (5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'). The PCR was performed in a total volume of 50 µl containing 1× HF buffer (New England BioLabs), 1 µl dNTP Mix (New England BioLabs), 1 U of Phusion® Hot Start II High-Fidelity DNA polymerase (New England BioLabs), 500 nM of the 27F-DegS primer, 500 nM of an equimolar mix of two reverse primers, 338R I and II. The size of the PCR products (~375 bp) was confirmed by gel electrophoresis using 5 µl of the amplification reaction mixture on a 1% (w/v) agarose gel. The PCR products were purified using QIAquick PCR Purification Kit (Qiagen). Then, the dual indices and Illumina sequencing adapters were attached using the Nextera XT index kit (Illumina) following the manufacture's instruction. Purified amplicon pools were 250 bp paired end sequenced using Illumina Miseq.