The open reading frame (ORF) sequence of CARHSP1 was searched using the UCSC and Ensemble genome browsers, and the ORF clone was amplified by PCR in order to append cloning sites to the 5′ and 3′ ends of the sequence using the following primers: sense, 5′-GAGGCGATCGCCATGTCATCTGAGCCTCCCCC-3′; antisense, 5′-GCGACGCGTGGAGCTGATGACATGTCCAGACCA-3′. The RNA was first isolated from THP-1 cells, and the full-length cDNA clone used as the template for CARHSP1 ORF cloning using the Phusion TM High-Fidelity PCR kit (NEB). The size of the amplification product was confirmed by agarose gel electrophoresis. The amplification product was digested using Sgf I and MIu I (NEB) and inserted into the Sgf I- and MIu I-cut pCMV6-AC-GFP vector (Origene, Rockville, MD, USA). The CARHSP1 overexpression plasmid sequences were confirmed by DNA sequencing, and the transfection efficiency was determined by qPCR. CARHSP1 siRNA was purchased from Santa Cruz Biotechnology, and 50 pmol/ml siRNA was transfected into THP-1 cells according to the manufacturer’s instructions.
Phusion high fidelity pcr kit
Manufactured by New England Biolabs
Sourced in United States, United Kingdom
The Phusion High-Fidelity PCR kit is a DNA polymerase-based solution for performing high-fidelity polymerase chain reaction (PCR) amplification. The kit includes a high-fidelity DNA polymerase, buffer, and dNTPs necessary for conducting PCR experiments.
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Lab products found in correlation
Lr clonase 2 enzyme mix, by Thermo Fisher Scientific (5 mentions)
Qiaquick pcr purification kit, by Qiagen (5 mentions)
Qiaamp viral rna mini kit, by Qiagen (4 mentions)
Superscript 3, by Thermo Fisher Scientific (3 mentions)
Phusion high fidelity pcr kit, by Thermo Fisher Scientific (3 mentions)
Dneasy blood tissue kit, by Qiagen (3 mentions)
Gibson assembly master mix, by New England Biolabs (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
T4 dna ligase, by Takara Bio (3 mentions)
Pcdna3.1 directional topo expression kit, by Thermo Fisher Scientific (3 mentions)
T4 dna ligase, by New England Biolabs (3 mentions)
Wizard sv gel and pcr clean up system, by Promega (3 mentions)
Purelink microbiome dna purification kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Pcr purification kit, by Qiagen (2 mentions)
Fetal bovine serum (fbs), by Gemini Bio (2 mentions)
Miseq platform, by Illumina (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
82 protocols using phusion high fidelity pcr kit
1
CARHSP1 Cloning and Overexpression Protocol
2
Characterizing the miR-155 Promoter Regulation
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The human miR-155 promoter region containing NF-κB p65 binding sites, extending from –1 bp to –2000 bp relative to the transcription start site, was generated by PCR using the following primers: sense, 5′-TATCTAGAGCGAGTTATATTGGCTGGGGTGG-3′; antisense, 5′-ATGCGGCCGC TGTGATACAAGCAATGGAGGT-3′. The genomic DNA of THP-1 cells was isolated for use as template. The purified fragment was digested using XbaI and NotI and inserted into the XbaI- and NotI-cut pRL-TK luciferase reporter vector. The promoter region was sequenced and compared with sequences in Genbank. The miR-155 promoters mutated at the p65 binding site 1 (–1108 bp to –1099 bp), site 2 (–978 bp to –969 bp), and site 3 (–714 bp to –704 bp) were generated by site-directed mutagenesis using a special polymerase contained in the PhusionTM High-Fidelity PCR kit (NEB).
3
Chicken Cecal Microbiome Analysis via 16S rRNA Sequencing
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All the samples collected during the chicken experiments (n = 58) were selected for microbiota analysis. Genomic DNA was extracted from 150 to 200 mg of cecal contents using the PureLink Microbiome DNA Purification Kit (Life Technologies, Invitrogen Corp.) and combined with RNAse treatment (10 units/h). After quality control with nanodrop, the 16S rRNA V4-V5 variable region was amplified, purified, and sequenced. Amplicon libraries were prepared by using Phusion® High-Fidelity PCR Kit (New England Biolabs Inc., Ipswich, MA, United States), as previously described (Kumar, 2015 ; Deblais et al., 2018 (link), 2019 (link); Kumar et al., 2018 (link); Srivastava et al., 2020 (link)). PCR products were cleaned using AMPure XP PCR (Beckman Coulter Inc., Beverly MA, USA) and were sequenced using Illumina MiSeq 300-base paired-end kit at the Molecular and Cellular Imaging Center.1 Sequencing raw data files are publicly available at NCBI Bioproject #PRJNA1023035.
4
Transient Expression of GFP-IgBBX in Tobacco
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The p35S::GFP-IgBBX constructs were introduced into tobacco epidermal cells via the Agrobacterium-infiltrated tobacco (Nicotiana benthamiana) leaf method [26 (link)]. The plasmid of IgBBX for transient transformation was generated using the Invitrogen Gateway system, according to the manufacturer’s instructions. The IgBBX ORFs, lacking the stop codon, were amplified using a Phusion®High-Fidelity PCR kit (New England Biolabs, Ipswich, MA, USA) (primers listed in Table S1 ), then inserted into the pMD19-T easy vector (TaKaRa, Tokyo, Japan) to allow its sequencing-based validation. Each pENTRTM1A-IgBBX plasmid was previously subjected to the LR Clonase™ II enzyme mix (Invitrogen, Carlsbad, CA, USA) reaction to obtain GFP-fused constructs using the binary vector pMDC43, resulting in the plasmid p35S::GFP-IgBBX. For transient expression, Agrobacterium tumefaciens strain EHA105 carrying the pMDC43 and pMDC43-IgBBX was grown separately to OD600 = 0.8 and coinfiltrated with the p19 strain into leaves of five-week-old N. benthamiana. The YFP fluorescent signals were monitored 48 to 72 h after infiltration using a confocal laser scanning microscopy (LSM 700, Carl Zeiss).
5
Conditional Mutants in Yeast
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S. cerevisiae strains used in this study are listed in Supplementary Table S6 , primers are listed in Supplementary Table S7 , and plasmids in Supplementary Table S8 . To create the Sdo1ts strain, the conditional TS18 intein28 (link),40 (link) was amplified by PCR from plasmid pS5DH-G4MINT (gift from N. Perrimon) and inserted between the SDO1 codons for K73 and C74 by homologous recombination. For the generation of Tif6-GFP mutants, site-directed mutagenesis of the pTIF6-GFP plasmid was performed using the Phusion High-Fidelity PCR kit (NEB) and transformed into XL1-Blue Electroporation-Competent cells (Agilent).
6
RNA Extraction and cDNA Synthesis
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Traditional the RNA purification method using TRIzol reagent (GibcoBRL, USA) was performed for tissue samples according to Rio et al. (2010) (link). Removal of genomic DNA from RNA samples was performed by DNase I (Thermo Fisher Scientific, USA) for 30 min at +37 °C as suggested by the manufacturer. RNA was purified and concentrated by phenol/chloroform/isoamyl alcohol extraction followed by subsequent ethanol precipitation. For cDNA preparation, the SuperScript III (Thermo Fisher Scientific, USA) Reverse Transcription kit was used as suggested by the manufacturer. For transcription analysis, RT-PCR was carried out by using Phusion High-Fidelity PCR Kit (New England BioLabs, USA) with corresponding F and R primers (see supplementary table S2, nos. 1–4, and fig. S6, Supplementary Material online). The RT-PCR program: denaturation for 1 min at 98 °C, 35 amplification cycles, each consisting of denaturation at 98 °C for 10 s, annealing at 63 °C for 15 s, primer extension at 72 °C for 10 s, and final elongation at 72 °C for 5 min. As a negative control, we used the same amount of RNA treated with DNase I not reverse-transcribed.
7
Isolation and sequencing of barley MTOPVIB
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Total RNA was isolated from 100 mg of barley anthers using Trizol (Invitrogen). One µg of RNA was employed for cDNA synthesis using the Invitrogen FirstStrand cDNA Synthesis Using SuperScript II RT kit following manufacturer’s instructions. cDNA samples were used for PCR amplification using the NEB Phusion High Fidelity PCR Kit and Sanger resequencing of the coding sequence of barley MTOPVIB. For primer sequences see Online resource 1.
8
Viral RNA Sequencing via SMRT
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Top eight samples were taken to perform single-molecule real-time sequencing (SMRT). The cDNAs were synthesized from viral RNAs by reverse transcription using Uni12 and Uni13 primers (Bi et al., 2016 (link)). The PCR was performed using a Phusion High-Fidelity PCR Kit (New England Biolabs) utilizing the barcoded influenza A virus general primers (Supplementary Table S1 ) (Mei et al., 2016 (link)). The concentration of PCR product was quantified by the Agilent Technologies 2,100 bioanalyzer. The two corresponding volumes of PCR products (containing equal mass of dsDNA) were mixed into one sample and quantified in the bioanalyzer again. About 2–3 μg mixed sample was used to SMRTbell library construction following the 2 kb template preparation protocol (Roberts et al., 2013b (link)). The sequencing was performed on a PacBio RS II instrument (Pacific Biosciences, USA) with one SMRT Cell used for each library, using P6/C4 chemistry with a 4 h movie (Bull et al., 2016 (link)). SMRTbell adapter sequences were removed and circular consensus sequence (CCS) reads were achieved with SMRT Analysis v2.3 (Roberts et al., 2013a ).
9
LMNA Variant Identification via iPSC DNA
10
RNA Extraction and Splicing Profiling
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RNA was extracted using standard Trizol RNA extraction. cDNA was synthesized from 500 ng total RNA in a 10 μl reaction, using Superscript VILO cDNA synthesis kit (Invitrogen) following manufacturer’s instructions. Splicing profiles were monitored by PCR using primers in flanking exons. For each PCR, 1 μl diluted cDNA (1/8) was used as template in a 10 μl PCR reaction using Phusion High-Fidelity PCR Kit (NEB, UK) following manufacturer’s instructions. Splicing profiles were monitored and quantified using the Qiaxcel capillary electrophoresis system (Qiagen) and PSI was calculated as described previously18 (link). All primers used for splicing assays are provided in Supplementary Data 3 .
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