RNA-seq of stool 14
Total DNA was extracted from 200 mg of collected feces per sample usinga DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNAconcentrations were measured using a Victor Nivo (PerkinElmer )). 16S rRNA amplicons were preparedwith a V3 V4 targeting universal primer set ( 341 F 805R ) under the following PCR condition: Thecycle condition for 1st PCR was 3 min at 95C for heat activation, and 25 cycles of 30 sec at 95C,30 sec at 55C and 30 sec at 72C, followed by a 5 min final extension at 72C The 1st PCR productwas purified with AMPure beads (Agencourt Bioscience, Beverly, MA). Following purification, the10ul of 1st PCR product was PCR amplified for final library construction containing the index usingNexteraXT Indexed Primer. The cycle cond ition for 2nd PCR was same as the 1st PCR conditionexcept for 10 cycles. The PCR product was purified with AMPure beads. The final purified productis then quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA LibraryQuantificato in kits for Illumina Sequecing platforms) and qualified using the TapeStation D1000ScreenTape (Agilent Technologies, Waldbronn, Germany). And then we sequenced using the MiSeqplatform (Illumina, San Diego, USA)