Biorad s100 gradient thermal cycler

Bacterial DNA from sediment samples was extracted using DNeasy Power Soil Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. DNA quantity was measured using NanoDrop Spectrophotometer (Thermo Fisher Scientific, USA), followed by dilution to 50 ng/μl final concentration. A total of 35 cycles of amplification for 50 µl final reactions were conducted in a BioRad S100 Gradient Thermal Cycler (Bio-Rad Laboratories, Inc., Foster City, California, USA) containing 2 µl template DNA, 1 µl of each V3-V4 sequencing primers (Part # 15,044,223 Rev. B), 25 µl of Hot Start Taq 2X Master Mix (New England BioLab Inc., USA) and 21 µl DEPC treated water. AMPure XP beads were used to process and clean amplified PCR products, and then, in accordance with the Illumina standard methodology (Part # 15044223 Rev. B), amplicon barcoding was performed using a secondary PCR. Samples were sequenced up to 50,000 reads on an Illumina MiSeq platform (Illumina Inc., USA) using a v3 kit (600 cycles, Part # MS-102-3003).

The bacterial DNA from pooled samples was extracted using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. After quantification in NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), extracted DNA was diluted into 50 ng/μl final concentration for PCR. Fifty microliters of PCR master mix was prepared by mixing 25 µL Hot Start 2X Master Mix (New England BioLab Inc., Lawrenceville, GA, USA), 2 µL of respective template DNA, 1 µL of each V3 and V4 sequencing primers (Part # 15044223 Rev. B) and 21 µL of DEPC treated water (Sigma-Aldrich, Germany). Forty cycles of amplification reactions were then performed in a BioRad S100 Gradient Thermal Cycler (Bio-Rad Laboratories, Inc., Foster City, California, USA). After visualization of PCR products in 1% agarose gel and subsequent clean-up with beads, each PCR amplicon was barcoded via a secondary PCR according to the Illumina standard protocol (Part # 15044223 Rev. B). Each sample was then sequenced up to 40,000 reads on an Illumina MiSeq platforms (Illumina Inc., San Diego, California, USA) at Harry Perkins Institute of Medical Research, Western Australia, using a v3 kit (600 cycles, Part # MS-102-3003).

At the end of the experiment, gut samples prepared as described in marron sampling were used for DNA extraction. Due to special role in digestion, absorption and immunity, hindgut was selected for 16S rRNA sequencing (Wang et al., 2018 (link)). The bacterial DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Crawley, UK) following manufacturer’s instructions. Extracted DNA was checked for quantity in a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and diluted to 30 ng/µL as final concentration. Thirty cycles of PCR amplification were performed in a BioRad S100 Gradient Thermal Cycler (Bio-Rad Laboratories, Inc., Foster City, CA, USA) with V3–V4 sequencing primers (Part # 15044223 Rev. B) and Hot Start Taq 2X Master Mix (BioLab Inc., Lawrenceville, GA, USA). Amplified PCR products were separated by 1% agarose gel and visualized under a gel documentation system (FujiFilm LAS–4000 Image Analyzer, Boston Inc., Foster City, CA, USA). Secondary PCR was applied for the barcoding of 16S rRNA PCR amplicon of each sample according to the Illumina standard protocol (Part # 15044223 Rev. B). The samples were then sequenced up to 20,000 reads on an Illumina MiSeq platforms (Illumina Inc., San Diego, CA, USA) at Harry Perkins Institute of Medical Research, Western Australia, using a v3 kit (600 cycles, Part # MS-102-3003).