Nucleobond axg 20 columns

The extraction of genomic DNA was performed using Nucleobond^® AXG 20 columns with a Nucleobond Buffer set III kit (Macherey Nagel, Hoerdt, France) according to the manufacturer’s instructions for Illumina sequencing, and using an increased initial culture volume of 10 mL for Oxford Nanopore sequencing to increase the DNA concentration. A NucleoSpin^® Tissue kit (Macherey Nagel) was used for DNA extraction for conventional PCR.

After a centrifugation step of 150 mL cultures of each strain, DNA extractions were performed on the pellets using the ZymoBIOMICS DNA mini kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocol. The Macherey Nagel genomic DNA and total RNA purification kit with NucleoBond AXG20 columns and buffer set III (Macherey-Nagel, PA, USA) was employed for PMC 881.14 and PMC 884.14. Metagenome sequencing was performed by GenoScreen (GenoScreen, Lille, France) using the Nextera XT DNA sample preparation kit (Illumina) for 2 × 250 bp and using a SMRT2 cell (PacBio) Illumina and Pacbio raw reads were corrected using SPAdes 3.12 and Canu 1.8, respectively, before the assembly performed with Unicycler hybrid-assembler, with default parameters [28 (link),29 (link),30 (link)]. For each new assembly, scaffolds were binned using MyCC (k-mer size = 4, minimal sequence size = 1000) and taxonomically annotated using CAT [31 (link)]. Congruent data between both methodologies allowed to characterize the cyanobacteria draft genomes.

M. hominis total genomic DNA extractions were performed using 10 mL cultures with NucleoBond^® AXG20 columns and the NucleoBond^® Buffer Set III from Macherey Nagel according to the manufacturer’s instructions. Transposon insertion sites were then determined by single-primer PCR. The 25 µL final reaction volume contained 1X PCR Buffer (Promega), 3 mM MgCl₂, 1 µM of SG9 primer (5′-TTTGGTTCAGAAACTGGTGCT-3′), 0.2 mM dNTPs, 0.1 µL of HotStart G2 DNA polymerase (Promega), and 2.5 µL of transformant DNA. The PCR amplification cycle was performed as previously described^{53 (link)}. The insertion positions were determined by Sanger sequencing (Eurofins genomics) of the PCR products with the nested primer MT85-1 (5′-ACAGTAATTGCGGGTGGATC-3′).