title: RNA-Seq Rat  day14
design: After euthanizing, fecal samples were collected from the colon and dispatched to Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China, for microflora analysis, according to the manufacturer's guidelines, FastPure Stool DNA Isolation Kit (MJYH, shanghai, China) was used to obtain microbial genomic DNA from Wistar rat fecal samples. The 1.0% agarose gel electrophoresis and a NanoDrop ND-2000 spectrophotometer (Thermo Scientific Inc., USA) were used to analyze the quality and concentration of DNA and preserved at -80  for later use. The hypervariable region V3-V4 of the bacterial 16S rRNA gene was obtained using primer pairs 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') and (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2R    (5'GCTGCGTTCTTCATCGATGC-3') primers pair were used to obtained ITS region of fungi ITS rRNA gene T100 Thermal Cycler (BIO-RAD, USA). The PCR set up solution counting 2L 2.5 mM dNTPs, 0.8 L each primer (5 M), 10 ng of template DNA, 4 L 5  Fast Pfu buffer, 0.4 L Fast Pfu polymerase, and ddH2O to make a final mixture of 20 L. The PCR reaction was run as follows: 3 min, initial denaturation at 95 , followed by twenty-seven PCR cycles of denaturing for 30 s, at 95 , 30 s for annealing at 55  and 45 s for extension at 72 and for 10 min, single extension at 72 . Each sample was amplified in triplicate. The amplicons were obtained from 2% agarose gel, and Synergy HTX (Biotek, USA) was used for quantification and purification. The Illumina MiSeq sequencing platform (Illumina, San Diego, CA, USA) was used to sequence the purified amplicons of ITS and 16s. ITS and 16S rRNA microbiota sequencing results were executed by the Majorbio Cloud Platform (www.majorbio.com).
identifiers.namespace: SUB14896073
identifiers.id: D14R