RNA-seq for Gut microbiota (mixed species)SD rats
Total RNA from diverse microbial groups was extracted via the CTAB method, with extraction efficiency assessed via agar gel electrophoresis and RNA content determined via UV spectroscopy. The PCR products were purified via AMPureXT beads, quantified via a Qubit, identified via 2% agarose gel electrophoresis, and recovered with the same beads. The purified products were evaluated via an Agilent 2100 Bioanalyzer and an Illumina Kapa kit, with libraries 2 nM selected. Qualified libraries (with unique indices) were pooled proportionally, denatured to single strands with sodium hydroxide, and sequenced on a NovaSeq 6000 (2250 bp paired-end) using a 512-cycle SP reaction box as a control. The data were subjected to demultiplexing, splicing, filtering, diversity analysis, species annotation, differential analysis, and advanced analyses.