title: ITS PCR amplification of different Prochlorococcus clades
design: PCR amplification of the ITS was carried out with specific primers for Prochlorococcus CS1_16S_1247F (5'-ACACTGACGACATGGTTCTACACGTACTACAATGCTACGG) and Cs2_ITS_Ar (5'-TACGGTAGCAGAGACTTGGTCTGGACCTCACCCTTATCAGGG)21,22. The first PCR was performed in triplicate in a total volume of 25 l containing 0.5 ng of template, 12.5 l of MyTaq Red Mix (Bioline) and 0.5 l of 10 M of each primer. The amplification conditions comprised steps at 95C for 5 min, 28/25 (16S/ITS) cycles at 95C for 30 sec, 50C for 30 sec and 72C for 1 min followed by one step of 5 min at 72C. All PCR products were validated on a 1% agarose gel and triplicates were pooled.  Subsequently, a second PCR amplification was performed to prepare libraries. These were pooled and after a quality control sequenced (2x250 paired-end reads) using an Illumina MiSeq sequencer