Nextera xt index kit 96

After visualization on the agarose gel, the LR-PCR fragments obtained were purified with Exo-Sap (Affymetrix, Santa Clara, USA) before they were quantified using Qubit dsDNA BR Assay System (Invitrogen, Carlsbad, USA) on the Qubit® 2.0 Fluorometer (Thermo Fisher, Waltham, USA).
In the next step, 27 amplicons of each patient were pooled in equimolar ratios. According to the manufacturer’s protocol, 1 ng of the pooled DNA fragments was subjected to the Nextera XT procedure (Illumina) using transposome technology. Finally, using Nextera XT DNA Sample Preparation Kit (Illumina) and Nextera^® XT Index Kit (96) (Illumina), we obtained eighty-seven both-side indexed DNA libraries ready for high-throughput sequencing. Quality control of the libraries was performed on TapeStation 2200 Instrument (Agilent, Santa Clara, USA) using an HS D1000 ScreenTape (Agilent). Normalization of all libraries was carried out with magnetic beads, according to the Nextera XT procedure.

DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany) following the manufacturer’s protocol. Amplification of the 16S V3-V4 region was performed using the universal primers reported previously (Klindworth et al., 2013 (link)). Samples were amplified using 2× KAPA HiFi HotStart Ready Mix (KAPA Biosystems, MA, USA), and indexed using the Nextera XT 96 Index Kit (Illumina, CA, USA). Lastly, the library was sequenced on an Illumina MiSeq platform and 300 bp paired-end reads were generated.

A Nextera XT DNA Library preparation kit and Nextera XT 96-index kit was used to prepare a total of 32 Illumina-compatible sequencing libraries across three experiments from both the direct lysate-derived acDNA and mRNA-enriched acDNA according to manufacturer’s instructions (Illumina), except the final volume was adjusted to 25 µL rather than 50 µL. The concentration of Nextera libraries were determined using a Qubit dsDNA High-Sensitivity (HS) kit (Thermo Fisher Scientific) run on a Qubit 2.0 fluorimeter (Thermo Fisher Scientific), and average fragment length was calculated using 2100 Bioanalyzer (Agilent Technologies) or Fragment Analyser (Agilent Technologies) reports and libraries were normalised by dilution to 4 nM.
Sequencing of all 32 libraries (Supplementary Table 1) was performed on a HiSeq2500 (Illumina) at the Otago Genomics Facility, housed in the Department of Biochemistry at University of Otago, Dunedin, NZ. Each experiment (Supplementary Table 1) was sequenced at a different time. Arabidopsis samples were run across two lanes with a 2x150bp paired-end V2 Rapid sequencing kit and kiwifruit samples were run across five lanes with a 2x250bp paired-end V2 Rapid sequencing kit.