All coding exons of SMAD4 in SAS, OECM-1, HSC3, FaDu, SCC25, OC3, and OC4 cells were amplified by PCR and then sequenced using the ABI BigDye Terminator Cycle Sequencing kit on an ABI 3730xl DNA analyzer (Applied Biosystems). The variants with MAF > 1% in the 1000 Genomes Project and dbSNP137 were filtered.
Abi bigdye terminator cycle sequencing kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The ABI BigDye Terminator Cycle Sequencing Kit is a reagent kit used in DNA sequencing. The kit contains the necessary components for performing cycle sequencing, which is a method of determining the nucleotide sequence of DNA molecules.
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Lab products found in correlation
Abi 3730 dna analyzer, by Thermo Fisher Scientific (5 mentions)
Illustra exoprostar 1 step, by GE Healthcare (3 mentions)
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (3 mentions)
Ampure xp system, by Beckman Coulter (2 mentions)
Abi 3730 sequencer, by Thermo Fisher Scientific (1 mentions)
Abi 3730 dna sequencer, by Thermo Fisher Scientific (1 mentions)
Massarray system, by Labcorp (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Epitect bisulfite kit, by Qiagen (1 mentions)
Exosap it, by GE Healthcare (1 mentions)
Repli g midi kit, by Qiagen (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Isohair, by Nippon Gene (1 mentions)
L dna analyzer, by Thermo Fisher Scientific (1 mentions)
Zr fungal bacterial dna miniprep kit, by Zymo Research (1 mentions)
96 capillary 3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Sequencing analysis v5, by Thermo Fisher Scientific (1 mentions)
Exosap it, by Thermo Fisher Scientific (1 mentions)
Qiasimphony, by Qiagen (1 mentions)
18 protocols using abi bigdye terminator cycle sequencing kit
1
Comprehensive SMAD4 Exon Sequencing
2
Genetic Screening for Dystonia Variants
3
Quantitative Analysis of P2X7R CpG Methylation
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Genomic DNA was isolated from fresh tissue using the QIAamp DNA Mini kit (Qiagen China Co., Ltd.) according to the manufacturer's protocol. Bisulfite treatment of genomic DNA was performed using the EpiTect Bisulfite kit (Qiagen China Co., Ltd.) according to the manufacturer's protocol. The cytosine-phosphodiester-guanosine (CpG) island located in the +26/+573 nt region of P2X7R was determined using the Sequenom MassARRAY® system (Sequenom, San Diego, CA, USA) using the following primers: Forward, 5′-AGGAAGAGAGTATTTTTGTGTAGGTATTTGGGGG-3′ and reverse, 5′-CAGTAATACGACTCACTATAGGGAGAAGGCTACATAATAACAACCTCCCTCCCTAC-3′. The PCR products were directly sequenced using an ABI BigDye Terminator Cycle Sequencing kit (PE Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) on an ABI 3730 DNA sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. At the corresponding CpG site, the sequencing trace was read as fully or partially methylated (C) and unmethylated (q). Quantitative analysis of CpG methylation was performed using MassCLEAVE base-specific cleavage combined with matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry using EpiTyper software version 4.0 (Sequenom).
4
Molecular Typing of Mumps Virus
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For MuV detected from mumps cases in the Netherlands nucleic acids were extracted and the SH and MF-NCR region were amplified as described previously (Bodewes et al., 2020 (link)). sequences PCR-amplified products were purified with ExoSAP-IT (GE Healthcare). Sanger sequencing was subsequently performed at BaseClear (Leiden, the Netherlands). Spanish MuV cases were amplified according to published protocol (Royuela et al., 2011 (link)). Positive samples were purified by an enzymatic reaction using Illustra ExoProStar 1-Step (GE Health Care Life Science, Freiburg, Germany) and sequenced using the Sanger method with the ABI Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Branchburg, NJ) using the corresponding forward and reverse primers (Gavilán et al., 2022 (link)).
5
Identifying MDS-Specific Genome Alterations
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To identify MDS-specific genome alterations, whole-genome sequencing was conducted on MonoMAC, MDS and BM-MSCs (Fig. 1 ). Genomic DNA was extracted with the DNeasy Blood & Tissue Kit (QIAGEN) or ISOHAIR (NIPPON GENE). For whole-genome sequencing, the DNA samples were amplified with the REPLI-g Midi Kit (QIAGEN). Sequencing libraries were prepared from 1 μg of the amplified DNA according to the TruSeq DNA Sample Prep Guide (Illumina). The libraries were sequenced on an Illumina HiSeq 2000 with HiSeq control software (HCS) version 1.5 and Real-Time Analysis (RTA) software version 1.13. After sequencing, reads were mapped to the human reference genome (GRCh37/hg19) with decoy sequences (hs37d5) using BWA [10 (link)] with the default options. Then, variant calling was conducted using the GATK Unified Genotyper [11 (link)].![]()
Study design. Whole-genome sequencing conducted with MonoMAC, MDS and BM-MSCs samples to identify MDS-specific genome alterations. Sanger sequence-based validation analysis was conducted on all samples, including a nail sample
6
Physical Map Construction of F. velutipes
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To generate a physical map, the BAC library was prepared from a partial HindIII and EcoRI digest of high-molecular-weight genomic DNA from F. velutipes in the vector pBeloBAC11 using standard methods [12] . BAC clone DNA was extracted from a single colony by a standard alkaline lysis extraction method. Inserts of 7,680 BAC clones were sequenced from both ends using universal primers, an ABI 3730×l DNA analyzer, and an ABI BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). A physical map of F. velutipes, based on BAC clones, was constructed by high information content fingerprinting (HICF) analysis according to the manufacturer’s standard protocol (ABI SNaPShot Multiplex System). BAC clones were analyzed in terms of band sizes following restriction enzyme digestion by GeneMapper and Genoprofiler, which were then assembled into an FPC map by FPC V8. The summed length of 13 FPC contigs was 39,612 consensus bands (CB), very close to the sum of contig sequence lengths [13] (link).
7
Mitochondrial DNA Sequencing and Haplogroup Analysis
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mtDNA was extracted from the blood, coronary artery and gingival tissue samples using the standard protocol.[14 ] Complete mitochondrial genome was amplified using 24 sets of overlapping primers.[15 ] The amplicons were directly sequenced using the ABI BigDye Terminator cycle sequencing kit, (Applied Biosystems, Foster City, CA, USA) and analyzed using ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Complete mtDNA sequences were aligned with the revised Cambridge reference sequence (NC_012920)[15 ] using sequence analysis and auto assembler tools. All the mismatched nucleotide sequences were carefully noted, compared with controls and searched in the human mitochondrial genome databases such as MITOMAP (http://www.mitomap.org ) and mitochondrial database (mtDB) (http://www.genpat.uu.se/mtDB ) for their significance. Each mtDNA haplogroup was constructed to find out if anyone haplogroup was associated with both diseases.
8
Phylogenetic Analysis of Sequence Data
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PCR products were purified by enzymatic reaction Illustra ExoProStar 1-Step (GE Health Care Life Science, Freiburg, Germany) and sequenced with the ABI Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Branchburg, NJ, United States) using the corresponding forward and reverse primers. Sequences were edited using BioEdit v.7.2.5 [19 ] and aligned with MAFFT v.7 [18 (link)]. Haplotypes (a set of identical sequences) were identified using DNAsp v5 software [20 (link)]. Phylogenetic analysis was performed by maximum likelihood method with the RaxML programme [21 (link)] through the BlackBox website (http://phylobench.vital-it.ch/raxml-bb ). To confirm the results, the PhyML 3.0 programme [22 (link)] was used through the ATGC portal (http://atgc.lirmm.fr/phyml/ ). The best evolutionary model was previously selected using jModelTest v.2.1.10 [23 (link)] according to the Akaike information criterion. Sequence fragments of the various genes were concatenated using Seaview v.4 [24 (link)] and in this case PartitionFinder v. 1.1.1 [25 (link)] was used to select the evolutionary model. Phylogenetic trees were edited using MEGA v.6.06 software [26 (link)].
9
Fungal Nuclear DNA Isolation and ITS Sequencing
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Isolation of nuclear DNA was performed by scraping fungal spores and hyphae from fungal isolates grown for ∼48 h at 30 °C on yeast malt extract agar plates(16 g l−1 agar, 3 g l−1 yeast extract, 3 g l−1 malt extract, 5 g l−1 peptone and 10 g l−1 glucose). Isolation of DNA was achieved by genomic DNA extraction using the ZR Fungal/Bacterial DNA MiniPrep kit (Zymo Research).
PCR was performed for the internal transcribed spacer (ITS) regions using fungal primers ITS-4 (5′-TCCTCCGCTTATTGATATGS-3′) and ITS-5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′)50 . Sequences were obtained with an ABI BigDye Terminator Cycle sequencing kit (Applied Biosystems) and resulting sequences were analysed using Geneious version 6 created by Biomatters (http://www.geneious.com ) and searched against the available yeast sequences in GenBank using BLAST51 (link).
PCR was performed for the internal transcribed spacer (ITS) regions using fungal primers ITS-4 (5′-TCCTCCGCTTATTGATATGS-3′) and ITS-5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′)50 . Sequences were obtained with an ABI BigDye Terminator Cycle sequencing kit (Applied Biosystems) and resulting sequences were analysed using Geneious version 6 created by Biomatters (
10
Sanger Sequencing Workflow for Mutation Validation
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Five lines were randomly selected to verify the mutations that were identified bioinformatically with Sanger sequencing (Supporting Information, Table S1 ). Primers were designed using Primer3 (Rozen and Skaletsky 1999 ) and PCR products destined for sequencing were cleaned using a standard Exo-SAP protocol (Bell 2008 (link)), and sequenced with an ABI BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Completed sequencing reactions were submitted to the Georgia Genomics Facility, and analyzed using an Applied Biosystems 3730xl 96-capillary DNA Analyzer.
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