Qiaquick pcr purification kit

Table S3 provides details for the oligonucleotides and processing steps used for every DNA template preparation used in this work. Unmodified DNA templates were PCR amplified from plasmid DNA using Vent (exo-) DNA polymerase and purified by agarose gel extraction exactly as described previously (39 (link)). DNA templates that contained an internal etheno-dA transcription stall site were PCR amplified from an unmodified linear dsDNA using Q5 High-Fidelity DNA polymerase; the unmodified linear dsDNA that was used as a template for these reactions was PCR amplified from plasmid DNA using Vent (exo-) DNA polymerase and purified by agarose gel extraction. After PCR amplification, DNA containing an internal etheno-dA modification was purified using a QIAquick PCR Purification Kit (Qiagen), processed by translesion DNA synthesis using a mixture of Sulfolobus DNA Polymerase IV and Vent (exo-) DNA polymerase, and purified a second time using a QIAquick PCR Purification Kit; these steps were performed exactly as described previously (39 (link)). A step-by-step protocol describing this procedure is available (61 ).

The amplified product from the second LM-PCR was purified using a QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions with minor modifications. Final elution volume was 41.5 μL, of which 1.5 μL was used for quantification by absorbance measurement. The rest was divided in two separate 20 μL aliquots. Adaptors were removed by digestion for 16 h at 37°C with 1 U of MseI (New England Biolabs). The mixture also contained 1× bovine serum albumin (New England Biolabs, Ipswich, MA, USA), 1 × Buffer 4 (New England Biolabs), and 24 μL of nuclease-free water. The reaction was terminating by heating for 20 min at 65°C. The sample was then purified using a QIAquick PCR Purification Kit (Qiagen), eluted in 23.5 μL of nuclease-free water and quantified by absorbance.

The SPEX experiments generally followed a published protocol (Brotherton et al., 2007 (link)) using DNA extracts prior to Illumina library construction with three modifications: (1) Illumina sequencing adaptors were attached to the 5′ end of the primers used in the first round of partially nested PCR; (2) MyTaq HS Mix (Bioline, BIO-25045) was used instead of Platinum Taq DNA Polymerase High Fidelity in the first round of a partially nested PCR; (3) only one round of a partially nested PCR amplification was performed. The nested PCR products were then quantified by qPCR and indexed using Illumina indexing primers (Table S3). The indexed PCR products were purified using a QIAquick PCR Purification Kit (Qiagen). The amplicons were quantified by qPCR and subjected to a second round of amplification using the same conditions as the first round. The products were purified again using the QIAquick PCR Purification Kit (Qiagen), quantified by qPCR and pooled at equimolar ratios. All PEC and SPEX products were pooled and measured using High Sensitivity DNA chips on an Agilent 2100 Bioanalyzer, then sequenced at the National High-throughput DNA Sequencing Centre, Copenhagen, Denmark using Illumina MiSeq Reagent Kit v2 (300 cycle).

A 16S rRNA gene clone library was obtained from DNA extracted at PNS5 sampling point after 570 days of plant operation. DNA was amplified using primers 27f (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492r (5′-TACGGYTACCTTGTTACGACTT-3′) using Hot Start Taq98 (Lucigen, Italy). PCR reactions were performed with the following cycles: 2 min at 98°C, 38 cycles for 30 s at 98°C, 30 s at 58°C, 1 min at 72°C and final 15 min at 72°C. PCR products were purified using the QIAquick^® PCR purification kit (Qiagen, Milan, Italy). Cloning of PCR products was carried out using pGEM-T Easy Vector System (Promega, Italy) into Escherichia coli JM109 competent cells (Promega, Italy) according to the manufacturer’s instructions. Positive inserts were amplified from recombinant plasmids obtained from white colonies by PCR using the sequencing primers T7f (5′-TAATACGACTCACTATAGGG-3′) and M13r (5′-TCACACAGGAAACAGCTATGAC-3′). PCR amplicons of 1,465 bp length were purified using the QIAquick PCR purification kit (Qiagen, Milan, Italy) and sequenced with primers 530F (5′-GTGCCAGCMGCCGCCG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′). A total of 100 clones were screened by PCR and multiple sequence alignments were performed with ClustalW2 to check the similarity among the sequences. The 16S rRNA gene sequences were deposited in the GenBank database under accession numbers from MG251533 to MG251561.

High-throughput specificity profiling of unmodified, BNA^NC-modified and LNA-modified crRNAs was performed as previously described^{40 (link)}. Briefly, 200 nM of concatemeric pre-selection libraries were incubated with 1000 nM Cas9 and 1000 nM gRNA or 100 nM Cas9 and 100 nM gRNA in Cas9 cleavage buffer (NEB) for 20 min at 37 °C. Pre-selection libraries were also separately incubated with 2 U of BspMI (NEB) in NEBuffer 3.1 for 1 h at 37 °C. Cas9-digested and BspMI-digested library members were purified with the QiaQuick PCR Purification Kit (Qiagen) and ligated to 10 pmol adaptor1/2(#) (post-selection) or lib adapter 1/lib adapter 2 (pre-selection) (sequences in Supplementary Table 9) with 1000 U of T4 DNA Ligase (NEB) in NEB T4 DNA Ligase Reaction Buffer for 16 h at room temperature. Adapter ligated DNA was purified using the QiaQuick PCR Purification Kit (Qiagen) and PCR amplified for 19–24 cycles with Q5 Hot Start High-Fidelity DNA Polymerase (NEB) in Q5 Reaction Buffer using primers PE2 short/sel PCR (post-selection) or primers lib seq PCR/lib fwd PCR (pre-selection) (sequences in Supplementary Table 9). PCR products were gel purified and quantified using a Qubit 2.0 Fluorometer (ThermoFisher) and subject to single-read sequencing on an Illumina MiSeq. Pre-selection and post-selection sequencing data were analyzed as previously described^{40 (link)}.