title: MesocosmExpt2_2013
design: Amplification of the V3 and V4 hypervariable regions of the 16S rRNA gene was performed  using oligonucleotide primers (Klindworth et al., 2013) modified by the addition of Illumina Tags  (underlined): S-D-Bact-0341-b-S-17 (5′-ACGACGCTCTTCCGATCTCCTAC  GGGNGGCWGCAG-3′) and S-D-Bact-0785-a-A-21 (5′-CGTGTGCTCTTCCGATCTGACT  ACHVGGGTATCTA-3′), resulting in amplicons that contained a 16S rRNA gene region of ~440  bp. The HiFi HotStart PCR kit (KAPA Biosystems, Boston, MA, USA) was used with the  following protocol: 0.2 mM dNTPs, 40 ng of DNA template, 0.02 U/μl KAPA HiFi Taq, 0.2 μM of  each primer in a total volume of 50 μl. PCR conditions were: initial denaturation at 95°C for 5 min;  followed by 27 cycles of 95°C for 1 min, 56°C (1 min) and 72°C (1 min); final extension at 72°C (5  min). Amplified products were purified using Agencourt AMPure XP purification kit (protocol  000387v001; Beckman Coulter, Brea, CA, USA) and quantified using Qubit dsDNA High  Sensitivity Assay (Agilent Technologies, Palo Alto, CA, USA). Then a second round of PCR was  performed using TruSeq® RNA PCR Primers (RPI1-33; Illumina, San Diego, CA, USA) to attach  6-bp multiplexing indices and Illumina sequencing adapters using the following protocol: 0.2 mM  dNTPs, 2 ng of the purified PCR product, 0.02 U/μl KAPA HiFi Taq, 0.2 μM of each primer in a  total volume of 50 μl. PCR conditions were: initial denaturation at 95°C for 20 sec; 10 cycles of  95°C (20 sec), 55°C (20 sec) and 68°C (20 sec); final extension at 68°C (40 sec). Amplicons were  purified and quantified as above and run on the MiSeq Illumina sequencing platform, producing 2 ×  250 bp reads (Illumina, San Diego, CA, USA).