Agencourt ampure xp magnetic beads

PCR was carried out for each DNA sample as a single reaction containing the nine GR bisulfite primer sets. Primary reactions contained 10 ng bisulfite converted DNA, 1X Multiplex PCR Plus Master Mix (Qiagen), and 0.8 μM multiplex primer mix (each primer), composed of GR bisulfite sequencing forward and reverse primers, in a 25-μL reaction volume. PCR conditions were 95 °C for 15 min followed by 35 cycles of denaturing at 95 °C for 30 s, annealing at 60 °C for 90 s, and elongation at 72 °C for 90 s, followed by a final extension at 68 °C for 10 min. Products were purified with Agencourt AmpureXP magnetic beads (Beckman Coulter) and eluted in 20 μL of water. Secondary PCR for sample barcoding was performed in 50 μL reactions as described above for singleplex library preparation, using 10 μL purified primary multiplex PCR product as a template. Samples were quantified by Qubit dsDNA High Sensitivity fluorometric assay (Invitrogen), pooled at equal concentrations, and then purified with Agencourt AmpureXP magnetic beads (Beckman Coulter) followed by Ion Torrent sequencing.

The QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used to isolate the high-quality DNA from each sample. According to the manufacturer’s instructions, 1 μg of genomic DNA was fragmented by sonication to a mean size of approximately 250 bp and subsequently used for whole genome bisulfite sequencing (WGBS) library construction using an Acegen Bisulfite-Seq Library Prep Kit (Acegen, Shenzhen, GD, China). Briefly, fragmented DNA was end-repaired, 5′-phosphorylated, 3′-dA-tailed, and then ligated to methylated adapters. The methylated adapter-ligated DNAs were purified using 1× Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA) and subjected to bisulfite conversion with a ZYMO EZ DNA Methylation-Gold Kit (Zymo research, Irvine, CA, USA). The converted DNAs were then amplified using 25 μl HiFi HotStart U+ RM and 8-bp index primers with a final concentration of 1 μM each. The constructed WGBS libraries were then analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies, SantaClara, CA, USA), quantified with a Qubit fluorometer with Quant-iT dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA,USA), and finally sequenced on an Illumina Hiseq X ten sequencer (PE150 mode) (Illumina, San Diego, CA, USA).

cDNA library preparation and Illumina MiSeq sequencing were carried out as described earlier [17 (link)]. Briefly, a 200-bp fragment library ligated with bar-coded adapters was constructed for individual strains with a NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA). Purification of the cDNA library was carried out with Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA). The quality assessment of the purified cDNA libraries was performed using a 4150 TapeStation (Agilent Technologies). About 151 paired end reads were generated by nucleotide sequencing by using an Illumina MiSeq sequencer (Illumina, San Francisco, CA) and a MiSeq Reagent Kit v2 (Illumina). CLC Genomics Workbench v7.0.3 (CLC Bio, Tokyo, Japan) was used to analyse the data. Contigs that shared a percent nucleotide identity of 95% or less were assembled from the obtained sequence reads by de novo assembly. The complete genome sequence of GII.17 strain (NICED-BCH-11889) was additionally determined by 5'-RACE [18 (link)]. Near-full genome sequences were deposited in DDBJ under the following accession numbers: LC769681.1–LC769715.1.

During pseudo-steady state, broth samples were taken from the enrichment culture, directly frozen in liquid nitrogen and subsequently stored at −80°C. Five hundred microliter samples were thawed on ice, pelleted by centrifugation (21,000 × g, 2 min, 4°C) and used for total RNA extraction with the RNeasy PowerMicrobiome Kit (Qiagen, Hilden, Germany), following the manufacturer’s instruction with the addition of phenol:chloroform:isoamy alcohol (25:25:1) and β-mercaptoethanol (10 μL mL^–1 final concentration). Cell lysis was with a FastPrep-24 bead beater (MP Biomedicals, Fisher Scientific, Hampton, VA, United States, four successive cycles of 40 s at 6.0 m s^–1, 2 min incubation on ice between cycles). Total RNA extracts were subjected to DNase treatment to remove DNA contaminants by using the DNase Max Kit (Qiagen, Hilden, Germany) and further cleaned up and concentrated with the Agencourt AMpure XP magnetic beads (Beckman Coulter, Brea, CA, United States) before rRNA depletion. Integrity and quality of purified total RNA were assessed on a Tapestation 2200 (Agilent, Santa Clara, CA, United States) with the Agilent RNA screen-tapes (Agilent, Santa Clara, CA, United States) and the concentration was measured using Qubit RNA HS Assay Kit (Thermo Scientific Fisher, Waltham, MA, United States).