Illumina MiSeq paired end sequencing; 16S rRNA gene sequence analysis of feces samples from rats treated with paclitaxel or vehicle controls
16S rRNA gene sequence analysis of feces samples from rats treated with paclitaxel or vehicle controls
Rat fecal samples were collected sterilely at day 14 and day 28 after the initiation of paclitaxel or vehicle treatments and stored at −80°C. Fecal DNA were extracted with the QIAamp DNA Stool Mini Kit (QIAGEN) and sequenced using tagged amplicon spanning the V3 to V4 region of bacterial 16S rRNA of approximately 540 bp using the Illumina MiSeq sequencer. Sequencing libraries were prepared using Illumina's TruSeq Nano DNA LT Library Prep Kit.1) First perform sequence end repair on the above amplified product, use the End Repair Mix2 in the kit to remove the overhanging base at the 5' end of the DNA sequence, and at the same time add a phosphate group to fill in the missing base at the 3' end;2) Add an A base to the 3' end of the DNA sequence to prevent the DNA fragments from self-ligating and ensure that the target sequence can be connected to the sequencing adapter (there is a protruding T base at the 3' end of the sequencing adapter);3) Add a sequencing adapter containing a library-specific tag (i.e. Index sequence) to the 5' end of the sequence so that DNA molecules can be fixed on the Flow Cell;4) Use BECKMAN AMPure XP Beads to remove self-ligated adapter fragments through magnetic bead screening, and purify the library system after adding adapters;5) Perform PCR amplification of the above-mentioned adapter-connected DNA fragments to enrich the sequencing library template, and use BECKMAN AMPure XP Beads to purify the library enrichment product again;6) Perform final fragment selection and purification of the library through 2% agarose gel electrophoresis.