Equiphi29 dna polymerase

Collected single protist cells were washed several times in solution U and then transferred to water droplets containing 0.01% Tween 20 (Nacalai Tesque, Kyoto, Japan). Bacterial cells that leaked from the ruptured host cell were collected with a glass capillary attached to the micromanipulation system and subjected to WGA using Thermo Scientific EquiPhi29 DNA Polymerase. The reaction was performed at 10-µL scale at 45 °C for 3 h (see Supplementary Methods for details). PCR was conducted using primer sets specific to the respective 16S rRNA phylotypes (Table S2), to check for the presence of their genomes. The WGA samples containing the target were re-amplified using EquiPhi29 DNA Polymerase at 50-µL scale. The reaction was performed at 45 °C for 2 h.

Due to the limited amount of cells and the slow growth, a DNA extraction from strain KI4 did not resolve detectable DNA concentrations. Therefore, a PCR was conducted directly on cells from the culture. This was done with the universal forward primer 27F; 5′-AGAGTTTGATCMTGGCTCAG-3′ and a reverse primer specific for the Pleurocapsales OTU from Bonthond et al. (2020 (link), pleuro592R; 5′-CACTGCTTGCCAGAAGTTG-3′). The product was sequenced in both directions at the Institute of Clinical Molecular Biology in Kiel using an Applied Biosystems 3730xl DNA Analyzer and the sequence was deposited in Genbank (accession: MW113706).
To sequence the genome, we also transferred one to a few cells from the culture to two PCR tubes and used the EquiPhi29 DNA Polymerase (Thermo Fisher Scientific) for whole genome amplification, following the protocol of the manufacturer with the incubation step at 45 °C for one hour. Both samples and a blank were submitted to the Beijing Genomics Institute (BGI, Shenzhen, China) where library preparation was conducted and both samples were sequenced on the MGISEQ-2000 platform.

The multiply-primed RCA (modified after Dean et al., 2001 (link)) is divided into three sub-steps: annealing, isothermal amplification, and inactivation. For the annealing, a 0.468 µl solution of random N2(sN)2N2 DNA primers (ordered lyophilized and desalted; Sigma-Aldrich) and 10x phi29 DNA Polymerase Reaction Buffer/phi29 buffer (New England BioLabs) is dispensed to the 0.612 µl LCR via a contactless nanoliter-dispenser (I.DOT, DISPENDIX) so that the resulting 1.08 µl annealing mixture consists of 56.7% (v/v) LCR, 166.7 µM random hexamers, and 1.67x phi29 buffer. That annealing mixture is denatured for 3 min at 95°C and then cooled down to 4°C as quickly as possible. This is followed by the isothermal amplification, for which a 0.72 µl solution of dNTPs, BSA (New England BioLabs), EquiPhi29™ DNA polymerase (Thermo Scientific), inorganic E. coli pyrophosphatase (New England BioLabs), and DTT is dispensed to the 1.08 µl annealing mixture via the I. DOT. All of that results in the final 1.8 µl RCA mixture consisting of 34% (v/v) LCR, 100 µM N2(sN)2N2, 1 mM of each dNTP, 0.4 μg/μl BSA, 1x phi29 buffer, 0.36 U/µl EquiPhi29™ DNA polymerase, 0.004 U/µl inorganic E. coli pyrophosphatase, and 4 mM DTT. This 1.8 µl of RCA mixture is incubated for 3 h at 40°C, followed by inactivation for 10 min at 65°C.