Bacterial DNA was isolated from fresh overnight cultures of the libraries rMAGE1, rMAGE2, rMAGE3, rMAGE4, rMAGE5 and rMAGE5 control with the kit UltraClean® Microbial DNA Isolation Kit (Mo Bio, QIAGEN). Fractions of the isolated genomes were used as templates for PCR with the primers tirDSF and tirDSR and the obtained amplicons were purified and sent for sequencing with Illumina MiSeq set-up. Isolated genomic DNA from the libraries rMAGE5, rMAGE10, MACS1EHEC, MACS1CR, MACS1EPEC, MACS2EPEC, MACS3EPEC, MACS4EPEC and MACS5EPEC was used as template for a PCR with the primers tirDSF and tirDSR and the resulting amplicons were cleaned NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel) and sent for PacBio circular consensus sequencing (CCS). For screening and for sequence confirmation, colony PCR products of around 1060 bp were obtained with the oligonucleotides CheckSATir_F and CheckSATir_Rev using Q5DNApolymerase (New England Biolabs) and the program as follows: 30 s at 98 °C, 30 cycles (15 s at 98 °C, 45 s at 70 °C and 90 s at 72 °C) and for final extension 120 s at 72 °C. PCR products were cleaned with the kit NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel) and sent to Macrogen for sequencing with the oligonucleotide CDRI_Fwd. Results were used to estimate the frequency of mutations in the libraries.
Nucleospin gel and pcr clean up
Manufactured by Macherey-Nagel
Sourced in Germany, United States, France, Argentina
The NucleoSpin Gel and PCR Clean-up is a lab equipment product from Macherey-Nagel. It is designed for the purification of DNA fragments from agarose gels and the removal of primers, nucleotides, and salts from PCR and other enzymatic reactions.
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Lab products found in correlation
Qiaprep spin miniprep kit, by Qiagen (8 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (8 mentions)
Pgem t easy vector, by Promega (8 mentions)
Nucleospin tissue, by Macherey-Nagel (7 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Hotstartaq plus dna polymerase, by Qiagen (5 mentions)
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (5 mentions)
3730 dna analyzer, by Thermo Fisher Scientific (4 mentions)
Ez dna methylation gold kit, by Zymo Research (4 mentions)
Nucleospin rna clean up, by Macherey-Nagel (4 mentions)
T4 dna ligase, by New England Biolabs (4 mentions)
Nucleospin plasmid, by Macherey-Nagel (4 mentions)
Methyl primer express v1, by Thermo Fisher Scientific (4 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (4 mentions)
Pyromark q24 system, by Qiagen (3 mentions)
Pyromark analysis software, by Qiagen (3 mentions)
Dyeex 96 kit, by Qiagen (3 mentions)
Pyromark gold q24 reagent, by Qiagen (3 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (3 mentions)
229 protocols using nucleospin gel and pcr clean up
1
Bacterial DNA Isolation and Sequencing
2
Cloning of Rainbow Trout α7nAChR
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The cloning of rainbow trout α7nAChR was achieved by first analyzing the α7nAChR nucleotide and protein sequences available in public databases (The National Center for Biotechnology Information, NCBI, and Universal Protein Resource, UniProt). Alignments of α7nAChR sequences from different species were done using ClustalX v2.1 and degenerate primers were designed in conserved regions (Supplemental Table 1). Full length α7nAChR was cloned from brain using 3` and 5`-RACE (Life Technologies, USA) following manufacturer's instructions with gene specific primers (Supplemental Table 1). PCR products were isolated with Nucleospin Gel and PCR Clean-up (Macherey-Nagel, Germany) and cloned into pGEM-T Easy Vector (Promega) in Escherichia coli DH5α cells (Life Technologies) following manufacturer's instructions. Plasmid clones were isolated using the Nucleospin Plasmid QuickPure kit (Macherey-Nagel) and digested with EcoRI (Thermo Fisher Scientific).
The plasmids were sequenced using SP6 and T7 primers with the sequencing kit BigDye Terminator v3.1 Cycle Sequencing (Applied Biosystems, USA) in the sequencer 3 130xl Genetic Analyzer (Applied Biosystems).
The plasmids were sequenced using SP6 and T7 primers with the sequencing kit BigDye Terminator v3.1 Cycle Sequencing (Applied Biosystems, USA) in the sequencer 3 130xl Genetic Analyzer (Applied Biosystems).
3
Validating Heterogeneous Mutational Patterns
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In order to validate the heterogeneous mutational patterns, Sanger sequencing analysis of ASXL3, TP53 (exon 5 and 8), and SMAD4 (exon 2, 9 and 11) was done using the PyroMark PCR Kit (Qiagen). PCR products were purified using the NucleoSpin® Gel and PCR Clean-up (Machery-Nagel, Düren, Germany) and sequenced by dye terminator cycle sequencing (BigDye Terminator v1.1 Cycle Sequencing kit, Applied Biosystems, Darmstadt, Germany) with universal M13- or PCR Primers. The sequencing products were purified using the DyeEx 96 Kit (Qiagen) and analyzed on a Genetic Analyzer 3500 (Applied Biosystems). Pyrosequencing, using the PyroMark PCR Kit (Qiagen) and the PyroMark Gold Q24 Reagents (Qiagen), was done to detect SNPs in BRCA1, BRCA2, CDH1, CTNNB1, KRAS, MLH1, MUTYH, PIK3CA, POLE, and RNF43. The PyroMark Q24 System and PyroMark analysis software (both Qiagen) were used for analysis. To validate low frequent mutations in ARID1A, ARID1B, AKT1, CLOCK, FLT4, IKBKB, IKZF3, LRP1B, MAP2K4, MCM8, PAX5, PRRC2A, and TP53BP digital PCR were done using the ddPCR™ Supermix for Probes (No dUTP) and the QX200™ Droplet Digital™ PCR System (both Biorad) following the manufacturer’s instructions. The primer sequences used are listed in Additional file 2 : Table S3. Additional file 2 : Table S4 summarizes the validated mutations.
4
Characterizing HIV Proviral Integration Sites
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For the library preparation, 2.5 µg genomic DNA of CHO.S18 was digested overnight with the restriction enzymes BamHI, BglII, EcoRI, MfeI, and HindIII in a total volume of 100 µl. The digest was purified via NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL) and linkers were ligated to the DNA ends overnight with T4 DNA Ligase. After heat inactivation at 70 °C for 15 minutes, 1 µl of each ligation was used as the first round PCR template for amplification with one primer complementary to the linker DNA and one complementary to the 3’end LTR of the HIV provirus. The secondary PCR added the indices needed for sequencing on the MiSeq sequencer (Illumina). Methods are essentially as described (Genome Walker Kit, Clontech) using primers described in the supplements. For CHO.S18, HIV-1 LTR specific reads covering the proviral integration boundary for 2 locations were obtained.
5
Genomic DNA Extraction and Analysis
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We obtained and analyzed genomic DNA from cyiPSCs using the DNeasy® Blood & Tissue Kit (Qiagen, Hilden) and PCR, respectively. PCR was performed using KOD plus Neo (Toyobo). Electrophoresis was performed with 100 bp and 1 kbp DNA ladders (New England Biolabs, Ipswich). The PCR products were purified using NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel) and digested by three restriction enzymes: HincII, BtsXI and MfeI (New England Biolabs). Table 1 lists the primers used.
For direct sequencing of the PCR products, we obtained DNA fragments from an agarose gel using NucleoSpin Gel and PCR Clean-up. The fragments were analyzed using a Big Dye Terminator Kit (Thermo Fisher Scientific) and Applied Biosystems® 3500 × L (Thermo Fisher Scientific). The sequence data were analyzed using SnapGene® ver 4.0.8 (GSL Biotech LLC, Chicago).
For direct sequencing of the PCR products, we obtained DNA fragments from an agarose gel using NucleoSpin Gel and PCR Clean-up. The fragments were analyzed using a Big Dye Terminator Kit (Thermo Fisher Scientific) and Applied Biosystems® 3500 × L (Thermo Fisher Scientific). The sequence data were analyzed using SnapGene® ver 4.0.8 (GSL Biotech LLC, Chicago).
6
Complete Genome Amplification and Sequencing of PCV4
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The complete genomes of PCV4-positive samples were amplified using two primer sets (Supplementary Table S1 ). The PCR reactions were performed in 25 µl reaction mixtures containing 3 µl of extracted DNA, 0.5 µM of forward and reverse primers, and 12.5 µl of Q5® High-Fidelity 2 × master mix. The PCR thermal profile involved an initial denaturation of 98 °C for 30 s followed by 35 cycles of 98 °C for 10 s, 72 °C for 30 s, 72 °C for 50 s, and final extension at 72 °C for 2 min. The PCR products were purified using Nucleospin™ Gel and PCR clean-up (MACHEREY–NAGEL, Germany) and submitted for sequencing by a barcode-tagged sequencing platform (Celemic, Seoul, Korea). The obtained nucleotide sequences were further analyzed and assembled with SeqMan, and Editseq software v.5.03 (DNASTAR Inc., Madison, Wisconsin, USA), then deposited in GenBank under accession no. ON854861-ON854863.
7
PCR Amplification and Sequencing Protocol
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DNA template for PCR was prepared by whole cell boiled lysates as previously described23 (link). All PCR amplifications were performed using TopTaq™ Master Mix Kit (QIAGEN, Germantown, MD, USA) according to the manufacturer’s instruction. Primers used in this study are listed in Supplementary Table 1 . PCR products were separated on 1.5% agarose gel electrophoresis (Sigma-Aldrish®) in 1XTris-acetate/EDTA (TAE) buffer. The gels were stained in RedSafe™ Nucleic Acid Staining Solution (iNtRON Biotechnology, NJ, USA) and visualized using the Omega Fluor™ Gel Documentation System (APLEGEN™ Gel Company, CA, USA). The PCR products were purified using Nucleospin® Gel and PCR clean up (Macherey–Nagel, Düren, Germany) and submitted for DNA sequencing at First Base Laboratories (Selangor Darul Ehsan, Malaysia). The DNA sequences obtained were compared with the reference sequence available at GenBank Database using the Blast algorithm (http://www.ncbi.nlm.nih.gov ).
8
Plasmid Linearization and In Vitro Transcription
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Plasmids were linearized by restriction with Xho1 [New England Biolabs (Australia) Pty Ltd., Victoria, Australia], electrophoresed and purified (NucleoSpin® Gel and PCR Clean-up, Macherey-Nagel, Düren, Germany). RNA transcripts were generated by in vitro transcription according to manufacturer’s instructions (MEGAscript® T7 transcription kit, Invitrogen by Thermo Fisher Scientific Australia Pty Ltd., Victoria, Australia) and RNA concentration was determined with a Qubit 4 Fluorometer (Invitrogen by Thermo Fisher Scientific Australia Pty Ltd., Victoria, Australia).
9
Whole-Genome Sequencing of Camel CSN1S2 Gene
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For the whole camel CSN1S2 gene sequencing, we selected 30 test samples (20 dromedaries randomly chosen from each region and 10 Bactrians). For amplification and sequencing, we relied on the dromedary genome (GenBank ID: NC_044512 region 69170091..69184631) as a template and DNAasis-Max ver. 3.0 software (Hitachi) to design 30 different primers (Supplementary Table S1 ). All primers were initially tested by gradient PCR in a final volume of 15 µL to determine the optimal annealing temperature. The PCR reaction mix included 50 ng of genomic DNA, 1× PCR buffer (Promega), 2.5 mM of MgCl2, 200 µg dNTP, 1 pmol of each primer, and 0.75 U of GoTaq® Flexi DNA Polymerase (Promega). The thermal profiles consisted of an initial denaturation at 95 °C (3 min), followed by 35 cycles at 95 °C (45 s), with annealing temperatures and times specific to each amplicon, then 72 °C (2 min), and ending with final extension at 72 °C (5 min), using a T100 thermal cycler (Bio-Rad). After PCR, the products were purified using NucleoSpin Gel and PCR cleanup (Machery-Nagel), and sequencing was outsourced to Eurofins Genomics (Ebersberg, Germany), using TubeSeq Supreme service.
10
Precise Targeted Gene Integration
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To evaluate the precise targeted integration, the DNA sequence around the integration site was investigated. The junction regions of the target site on the host genome and introduced gene were amplified by PCR using the following primer pairs: acta1-for-Seq-Fw and mAG-Rv, mAG-Fw and acta1-for-Seq-Rv, or GAP43-for-Seq-FW and GAP43-for Seq-RV (Additional file 2 : Table S4), and KOD -plus- Neo DNA polymerase (Toyobo). The PCR conditions were as follows: one cycle at 94 °C for 2 min, followed by 35 cycles of 98 °C for 10 s, 58 °C for 30 s, and 68 °C for 1 min. The resulting PCR products were subjected to electrophoresis with a 1% agarose gel. PCR fragments predicted to contain the introduced gene were excised from the gel and purified using NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL). The purified fragments were sequenced using the primers acta1-for-Seq-Fw and acta1-for-Seq-Rv (for acta1) or GAP43-for-Seq-FW and GAP43-for-Seq-RV (for gap43) (Additional file 2 : Table S4).
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