title: MESO-SCOPE 16SITS23S from all metagenomes
design: PCR amplification was performed using 27-F (5 AGRGTTYGATYMTGGCTCAG 3) and ACMITS-3-R (5TCATCGCCTCTGTGTGCC 3) 37,38. Amplicons were cloned using a TOPO-TA cloning kit (ThermoFisher K4575J10). Competent cells were cultured on Lysogeny broth (LB)-medium with kanamycin. Individual colonies were picked and sub-cultured on LB-medium with ampicillin. Sub-cultures were then picked and incubated in a liquid LB broth for three days before plasmid extraction with a QIAprep Spin Miniprep Kit. Plasmids underwent PCR amplification using the M13 primers included in the TOPO-TA cloning kit. After amplification, the PCR product was sent to the Genewiz sequencing facility in South Plainfield, New Jersey, for Sanger sequence generation.