16SRNA human gut, fecal sample
he V3-V4 hypervariable region of the 16S rRNA gene was amplified from each sample, high fidelity PCR reactions were performed, 50 L of master mix composed of 13L RNAse/DNAse free water and 25 L Platinum SuperMix (Invitro- gen, Carlsbad, CA, USA) were mixed into individ- ual tubes. For the initial reaction, 100 ng of DNA was added to the reaction wells. Barcode primers (2 mM) from the primer were added to corre- sponding tubes. Two different PCRs were set up separately. First V3-V4 regions of the 16S rRNA gene were amplified by PCR using a barcode. Cy- cling conditions were 95C for 5 min, followed by 30 cycles at 95C for 1 min, 45C for 1 min, 72C for 30 s. Amplicons were purified by using the QIAquick PCR purification Kit (Qiagen, Valencia, CA), and were quantified using the Qubit system (Invitrogen, USA) according to the manufacturers specifications. Equimolar of each sample were pooled. Second PCR consists at Illumina sequenc- ing adapters, cycling conditions were 95C for 5 min, followed by 5 cycles at 95C for 1 min, 50C for 1 min, 72C for 1 min. PCR products were cleaned using Agencourt AMPure Beads PCR pu- rification kit (Beckman Coulter, MA) PCR prod- ucts were quantified using Qubit system (Invitro- gen, USA) according to the manufacturers speci- fications.