Mag bind total pure ngs beads

Plasmids encoding the TRAC TALEN contained a T7 promoter and a polyA sequence. The TALEN mRNA from the TRAC TALEN plasmid was produced by Trilink. Sequence targeted by the TRAC TALEN (17-bp recognition sites, upper case letters, separated by a 15-bp spacer): (TTC​CTC​CTA​CTC​ACC​ATc​agc​ctc​ctg​gtt​atG​GTA​CAG​GTA​AGA​GCA​A).
The TALEN mRNA from the CD52 TALEN plasmid was produced by Trilink. Sequence targeted by the CD52 TALEN (17-bp recognition sites, upper case letters, separated by a 15-bp spacer): (TTC​CTC​CTA​CTC​ACC​ATc​agc​ctc​ctg​gtt​atG​GTA​CAG​GTA​AGA​GCA​ACG​CCT​GGC​A).
Plasmids encoding TALE-BE T-25 and CD52 TALE-BE contained a T7 promoter and a polyA sequence (TALE-BE Sequence and target sequence in Supplementary info). Sequence verified plasmids were linearized with SapI (NEB) befor in vitro mRNA synthesis. mRNA was produced with NEB HiScribe™ T7 Quick High Yield RNA Synthesis Kit (NEB). The 5′capping reaction was performed with ScriptCap™ m7G Capping System (Cellscript). Antarctic Phosphatase (NEB) was used to treat the capped mRNA and the final cleanups was performed with Mag-Bind TotalPure NGS beads (Omega bio-tek) and Invitrogen DynaMag-2 Magnet (ThermoFisher).

The V2–V3 portion of the 16S rRNA was amplified, using the primer set F101-R534, with a different IonXpress barcode per sample attached to the reverse primer. PCR reactions were performed using the Kapa Library Amplification Kit (Kapa Biosystems, Wilmington, MA, United States) and BSA 400 ng/μl, under the following conditions: 5 min at 95°C, 30 s at 95°C, 30 s at 59°C, 45 s at 72°C, and a final elongation step at 72°C for 10 min. DNA after normalization was quantified with a Qubit^® 2.0 Fluorometer (Invitrogen, Carlsbad, CA, United States). The amplicon size was checked on a 2% agarose gel. The subsequent step of PCR purification was carried out using the Mag-Bind^® Total Pure NGS beads (OMEGA Bio-Tek, Norcoss, GA, United States), retaining fragments > 100 bp. Template preparation was performed by the Ion PGM Hi-Q View kit on the Ion OneTouch^TM 2 System (Life Technologies, Grand Island, NY, United States) and sequenced using the Ion PGM Hi-Q View sequencing kit (Life Technologies, Grand Island, NY, United States) with the Ion PGM^TM System technology. Negative controls, including a no-template control, were processed with the clinical samples (Campisciano et al., 2017 (link)).

Libraries were prepared for RNA sequencing according to the instructions of the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New Englands Biolabs, Ipswich, MA, USA), with the following exception. RNA samples were fragmented 7 min instead of 15 min to produce fragments longer than 100 bp. Instead of the adaptors provided with the kit, we used TruSeq compatible YIGA adaptors (see Table S2 in the supplemental material). Nucleic acid purification was performed using Mag-Bind TotalPure NGS beads (Omega Bio-Tek Inc, Norcross, GA, USA) following the recommended ratio in the NEBNext kit. Ligation products were subjected to PCR amplification using a reverse primer containing a unique 8 bp index for each sample (see Table S2 in the supplementary material). The concentration of nucleic acid in the libraries was measured using a Quant-iT PicoGreen dsDNA assay kit (Invitrogen, Carlsbad, CA, USA) and 1.13 ng of each sample were pooled in 80 µL of nuclease-free water. Quantity and quality of the final mix were confirmed with an Agilent 2100 Bioanalyzer before submitting to a Nextseq500 High Output 75 bp sequencing.

Total genomic DNA was extracted from the cecal content using the QIAamp® DNA Stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instruction. The concentration and integrity of bacterial DNA were determined by NanoDrop One (Thermo Fisher Scientific, Madison, WI, USA) and agarose gel electrophoresis, respectively. Amplification of the V3-V4 hypervariable regions of the 16S rRNA gene was carried out at the University of Hawaii at Manoa Advanced Studies in Genomics, Proteomics, and Bioinformatics core facility as outlined in the Illumina 16S Metagenomic Sequencing Library guideline (Illumina) with the following modification. Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Life Technologies Corporation, Grand Island, NY) was used to set up the PCR reaction, Mag-Bind Total Pure NGS beads (Omega Bio-Tek) were used for PCR Clean-Ups and 35 cycles were used in PCR. Finally, amplicons were normalized, pooled, and sequenced on the Illumina MIseq sequencer.

Total genomic DNA was extracted from the cecal content using the QIAamp® DNA Stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instruction. The extracted bacterial DNA concentration was determined using NanoDrop one (Thermo Fisher Scientific, Madison, WI) and stored at -20°C until further analysis. The V3 and V4 hypervariable regions of the 16S rRNA gene were amplified as outlined in Illumina 16S Metagenomic Sequencing Library guideline (Illumina) with the following modifications: Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Life Technologies Corporation, Grand Island, NY) was used to set up the PCR reaction, and Mag-Bind Total Pure NGS beads (Omega Bio-Tek) were used for PCR Clean-Ups, and 35 cycles were used in the PCR. Briefly, 16S rRNA sequencing involved the following steps: 1) 1^st stage PCR: Amplicon PCR, 2) PCR Clean-Up, 3) 2^nd Stage PCR: Index PCR, 4) 2^nd PCR Clean-Up 5) Library quantification, normalization, and pooling, and 6) Library denaturing and MiSeq sample loading. The specific sequence of 16S rRNA gene used for amplified were forward primer (5′-CCTACGGGNGGCWGCAG-3′) and reverse primer (5′GACTACHVGGGTATCTAATCC- 3′) [16 (link)]. Finally, amplicons were normalized and pooled, and subsequently sequenced on the Illumina MiSeq sequencer at the University of Hawaii at Manoa in Genomics, Proteomics, and Bioinformatics core facility.