A total of 96 patients were included in the gene expression analysis using TempO‐Seq, transcriptome sequencing of approximately 20,000 protein‐coding genes (BioSpyder Technologies Inc.). The sample preparation methods have been described previously.20 Briefly, TempO‐Seq is an extraction‐free method. Samples (4–16 mm2 from the FFPE section) were added to the lysis reagent and detector oligos (Dos) for the genes to be measured, which were ligatable after annealing. Next, proteinase K was added. Hybridization exonuclease was then added to destroy the unhybridized Dos, followed by a single polymerase chain reaction. The samples were barcoded and pooled into a sequencing library. The sequencing library was purified using a Clontech NucleoSpin Gel and PCR Cleanup Kit (Clontech). Sequencing was performed using Illumina NextSeq. Demultiplexed FASTQ files were set as the sequencer outputs.
Nucleospin gel and pcr clean up kit
Manufactured by Takara Bio
Sourced in United States, Japan
The NucleoSpin Gel and PCR Clean-up kit is a laboratory equipment product designed for the purification of DNA fragments from agarose gels or PCR reactions. It efficiently removes primers, nucleotides, and other reaction components.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (6 mentions)
Bigdye terminator version 3.1 cycle sequencing kit, by Thermo Fisher Scientific (5 mentions)
Firstchoice rlm race kit, by Thermo Fisher Scientific (4 mentions)
Kod polymerase, by Merck Group (4 mentions)
Smarter race 5 3 kit, by Takara Bio (4 mentions)
Cloneamp hifi pcr premix, by Takara Bio (4 mentions)
Primestar gxl dna polymerase, by Takara Bio (4 mentions)
Stellar competent cells, by Takara Bio (3 mentions)
In fusion hd cloning kit, by Takara Bio (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Dneasy blood tissue kit, by Qiagen (3 mentions)
Tks gflex dna polymerase, by Takara Bio (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Take3 microspot plate reader, by Agilent Technologies (2 mentions)
5 alpha electrocompetent e coli, by New England Biolabs (2 mentions)
Plasmid giga kit, by Qiagen (2 mentions)
Endofree plasmid giga kit, by Qiagen (2 mentions)
Qiaprep spin miniprep kit, by Qiagen (2 mentions)
In fusion hd cloning plus kit, by Takara Bio (2 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
92 protocols using nucleospin gel and pcr clean up kit
1
Transcriptome Profiling of FFPE Samples
2
5'RACE of Chimera-Treated Tumor RNA
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5′-RACE was performed using SMARTer RACE5′/3′ kit according to the manufacturer’s protocol. RNA (3 μg each) from tumors treated with different chimeras was reverse transcribed into cDNA containing a SMARTerIIA oligonucleotide adaptor. Nested PCR was performed to detect the cleavage sites. For EGFR siRNA analysis, outer PCR was first run with EGFR reverse primer (5′-GGGCAGGTGTCCTTGCACGT-3′) and forward Universal Primer A Mix (UPM) (5′-CTAATA CGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′); then inner PCR was performed with EGFR reverse primer (5′-GCACGGCGCCATGCAGGATTTCCTGT-3′) and forward primer Universal Primer Short (5′-CTAATACGACTCACTATAGGGC-3′). For survivin analysis, outer PCR was first performed with survivin reverse primer (5′-TGCTAAGGGGCCCACAGGAAGGCTGGT-3′) and forward primer: UPM; then inner PCR was performed with survivin reverse primer (5′-AGCCTTCCAGCTCCTT GAAGCA-3′) and forward primer Universal Primer Short. PCR products were separated with 2% agarose gel electrophoresis, and DNA was extracted from gel with NucleoSpin Gel and PCR Clean-Up kit (Clontech). The purified PCR products were sequenced to determine identity.
3
High-throughput CRISPR Screening Sequencing
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3.33 µg of the isolated genomic DNA was used to amplify the bar-coded amplicons in 39 Herculase II DNA polymerase (Agilent cat. 600679, Santa Clara, CA) reactions per sample (primers described in Supplementary Data 13 ). 5 µl amplicon or 1 µl diluted plasmid library was used as template in 13 50 µl Herculase II DNA polymerase reactions per sample to attach pooled variable-length spacers and Illumina indexes (primers described in Supplementary Data 13 ). 24 cycles were used to amplify DNA in the first and second PCR, respectively. The amplicon fragments after PCR 2 have the following sequence (354–362 bp library with variable 20 bp sgRNA sequence in the middle) (SF1). DNA was pooled by sample and purified using the Nucleospin Gel and PCR Clean-up kit (Clontech cat. 740609.250, Mountain View, CA). DNA was quantified using a Qubit high-sensitivity DNA quantification assay (Thermo Fisher cat. Q32851) and Take3 microspot plate reader (BioTek). DNA quality was analyzed by Experion CHIP assay (BioRad cat. 7007-163, Hercules, CA). Clusters were generated on the flow cell using the HiSeq Rapid Duo CBot Sample Loading Kit (Illumina CT- cat. 403-2001, San Diego, CA). A single-read rapid run of 75 cycles was performed on a HiSeq. 1500 (Illumina cat. GD-402-4002) using the HiSeq Rapid SBS kit (Illumina cat. FC-402-4022) with 10% PhiX.
4
PHACTR1 Transcript Identification via RACE
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We extracted total RNA from HUVEC, teloHAEC, HCAEC, HAEC, HCASMC and HASMC P20 (with a passage (P) lower than 8 for primary cells unless specified) cells grown to confluence in 100 mm dishes with the RNeasy Plus Mini Kit (Qiagen). For the RACE experiments, we also added RNA from one hCA donor (#ICM167), adult brain, and adult heart. RNA integrity and concentration were measured by Agilent RNA 6000 Nano II assays (Agilent Technologies) on an Agilent 2100 Bioanalyzer. We reverse transcribed 1 μg of total RNA using a modified oligo (dT) primer and the SMARTScribe Reverse Transcriptase (Clontech). We specifically amplified cDNA of PHACTR1 using the SMARTer RACE 5′/3’ Kit (Clontech). The gene specific primers are in Additional file 1 . The RACE products were purified by gel extraction with the NucleoSpin Gel and PCR Clean-Up Kit, cloned in pRACE vector with the In-Fusion HD Cloning Kit and transformed with Stellar Competent Cells (Clontech). After overnight incubation, plasmids were extracted from single bacterial colonies with QIAprep Spin miniprep kit (Qiagen). We sequenced PHACTR1 inserts by Sanger sequencing using M13 primers (Additional file 1 ).
5
Amplification of G. vaginalis tuf gene
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The primer pair Gvag_tuf_AS3/Gvag_tuf_S4 was used to amplify a 149-bp fragment from the tuf gene (GI:311114364) of G. vaginalis strain JCP8481B. The PCR product was purified using the Nucleospin Gel and PCR Clean-Up Kit (Clontech, Takara Bio, Mountain View, CA, USA) and cloned into the Zero Blunt TOPO vector (Invitrogen) according to the manufacturer's instructions.
6
Genome Editing Efficiency Analysis
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Genomic DNA was extracted using the PureLink Genomic DNA Mini Kit (Invitrogen). Specific genomic loci were amplified using Velocity DNA Polymerase (Bioline). Off-target loci represent the top predicted off-target sites in the CRISPR Design Tool (crispr.mit.edu )68 (link). PCR products were gel-extracted (NucleoSpin Gel and PCR Clean-up kit, Clontech) and sent for Sanger sequencing. Sequencing results could then be uploaded to the Synthego ICE Analysis tool (v3) allowing for inference of the percent indels in the sample. For deep sequencing, the gel-extracted products were pooled and prepared for sequencing via paired-end 2 × 150 bp iSeq (Illumina, San Diego, CA) sequencing in-house.
7
Bacterial Strain Cultivation and Plasmid Isolation
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All strains were grown in lysogeny broth (LB), Rich Defined Media (RDM, Teknova) or on LB agar, supplemented with 50 μg ml−1 kanamycin, 100 μg ml−1 carbenicillin or 25 μg ml−1 chloramphenicol (Sigma) for selection. Plasmids were isolated using a QIAQuick Miniprep kit (Qiagen). Polymerase chain reaction (PCR) reaction products were purified using a GeneJet Gel extraction kit (Thermo Scientific) or NucleoSpin Gel and PCR Clean-Up kit (ClonTech). Plasmids and PCR products were sequenced using Sanger sequencing (Elim Biopharma or MCLab). All PCR and cloning reactions were performed on a S1000 Thermal Cycler (Bio-Rad). Information about the E. coli strains used in this experiment can be found in the Supplementary Data .
8
RNAi-mediated gene silencing in Drosophila S2 cells
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PCR templates for in vitro transcription (IVT) were amplified from S2 cell cDNA or pGFP5(S65T) plasmid using Phusion Hot Start Flex DNA Polymerase (NEB) and primers listed in Supplementary Table 5 . PCR products were purified using NucleoSpin Gel and PCR Clean-Up Kit (Clontech). IVT was performed to generate dsRNA using the T7 High Yield RNA Synthesis Kit (NEB). Template DNA was removed using Turbo DNAse (Ambion) and dsRNA was purified using the NucleoSpin RNA Clean up XS kit (Clontech). To perform RNA interference (RNAi), S2 cells were seeded at a density of 1 million cells ml–1 of serum-free medium. As control RNAi, a total of 30 μg green fluorescent protein (GFP) dsRNA was added to cells. For Mettl3 RNAi, 15 μg Mettl3 dsRNA number 1 plus 15 μg Mettl3 dsRNA number 2 were added. After 6 h, medium was replaced with serum containing medium. Treatment with dsRNA was repeated after 48 and 96 h. Cells were collected after 120 h.
9
Defining LeXis Transcript Boundaries
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The 5' and 3' ends of the LeXis transcript were defined using mouse liver RNA and the FirstChoice RLM-RACE kit (Ambion) according to manufacturer's protocol, with modifications. Briefly, for the 5' RACE, degraded mRNA 5' ends were dephosphorylated with CIP and then full-length mRNA was decapped with TAP. Following 5'RACE adapter ligation, reverse transcription was performed using SuperScriptIII First-Strand Synthesis system (Invitrogen) and LeXis-specific primers. For the 3’RACE, the RNA was reverse transcribed using SuperScriptIII First-Strand Synthesis system (Invitrogen) and the adapter-linked oligo dTs. The resulting cDNA was amplified by nested PCR across a 55–65 °C melting temperature gradient using KOD polymerase (Millipore), with the inner primers containing attB sequences. Aliquots of reactions were inspected on 1% agarose gels for product size and abundance. Products of select PCR reactions were purified using NucleoSpin Gel and PCR Cleanup kit (Clontech) and were inserted into pDONR221 by Gateway cloning. Cloned fragments were sequenced and then aligned to the mouse genome with the BLAST analysis tool.
10
Genomic DNA Isolation and Genome Walking
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Genomic DNA was isolated from the 63–3 cell line and used for genome walking (Universal Genome Walker Kit; Clontech, Mountain View, CA). Briefly, four pools of adaptor-ligated genomic DNA were produced by restriction digestion and subsequent ligation with a single universal adaptor according to the manufacturer’s recommendation. Two sets of nested oligonucleotide primers were designed corresponding to opposite ends of the transgene construct and were oriented for the amplification of flanking genomic DNA (i.e., primers were designed from the minus strand for the 5’ end of the construct and plus strand for the 3’ end of the construct). An initial PCR amplification was performed using the innermost primers corresponding to each of the construct ends and a primer directed at universal adapter sequence. First round amplicons were used as template for a second round of amplification with the appropriate nested primer sets. PCR products were separated on a 1.2% agarose gel; bands of interest were extracted and purified using NucleoSpin Gel and PCR Clean-up kit (Clontech) and then sequenced. Genomic sequences were mapped to the pig genome using BLAST [34 (link)]
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