An amount of 2 μg of total RNA from each sample was reverse-transcribed and labeled directly with fluorescent dye. The 30-μl RT reaction mixture contained 5 μg of random primers, 40 U RNaseOUT™ Recombinant Ribonuclease Inhibitor, 6 μl 5x first-strand buffer, 3 μl of 100 mM DTT, 400 U SuperScript™ III Reverse Transcriptase (Invitrogen, Life Technologies Ltd, Paisley, UK), 0.6 μl dNTP mix (25 mM dATP, 25 mM dGTP, 25 mM dTTP, 10 mM dCTP [Promega Corporation, Madison, WI, USA]), and 2 nmol Cy3-dCTP or Cy5-dCTP (GE Healthcare, Buckinghamshire, UK). Each mixture was incubated for 3 h at 46°C and stopped with 1.5 μl of 20 mM EDTA. After addition of 15 μl of 0.1 M NaOH the RNA was hydrolyzed for 15 min at 70°C, followed by neutralization with 15 μl of 0.1 M HCl. The labeled cDNA was purified using a DNA purification column (QIAquick PCR purification kit; Qiagen) and eluted into 44 μl of elution buffer (Qiagen).
Cy3 dctp
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Belgium
Cy3-dCTP is a nucleotide analog used in various molecular biology techniques. It is a fluorescently labeled form of the deoxycytidine triphosphate (dCTP) nucleotide, with the Cy3 fluorescent dye attached. Cy3-dCTP can be incorporated into DNA or RNA during synthesis, allowing for the detection and visualization of the labeled molecules.
Automatically generated - may contain errors
Lab products found in correlation
Cy5 dctp, by GE Healthcare (19 mentions)
Bioprime array cgh genomic labeling system, by Thermo Fisher Scientific (4 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Feature extraction software 9, by Agilent Technologies (2 mentions)
32k mouse genome array, by CapitalBio (2 mentions)
Lux scan 3.0 scanner, by CapitalBio (2 mentions)
Lux scan 3.0 image analyzer, by CapitalBio (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Fluor x dctp, by GE Healthcare (2 mentions)
Yeast trna, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Elution buffer, by Qiagen (1 mentions)
Rnaseout recombinant ribonuclease inhibitor, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Random primer, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Genespring, by Agilent Technologies (1 mentions)
Human lncrna mrna array v4, by Agilent Technologies (1 mentions)
33 protocols using cy3 dctp
1
Fluorescent RNA Labeling and Purification
2
Transcriptome Analysis of Bacterial Mutants
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For each replicate, 2 μg of RNA was labeled with Cy3 dCTP (GE Healthcare) using random primers (Invitrogen) and Superscript II reverse transcriptase (Invitrogen). Fluorescently labeled cDNAs were purified and hybridized onto BμG@S SAv2.1.0 microarrays provided by the Bacterial Microarray Group at St. George's, University of London, using a one-color microarray-based gene expression system. Washing, scanning, and feature extraction procedures were performed as described previously (42 (link)). Array design is available in BμG@Sbase (accession number A-BUGS-42) and also ArrayExpress (accession number A-BUGS-42). Statistical analyses were performed using GeneSpring (Agilent Technologies). Differentially expressed genes were defined as those that showed >2-fold up- or down-regulation with respect to the wild type, with a p value of <0.05 determined by two-way analysis of variance with Benjamini and Hochberg false discovery rate correction. A total of 465 differentially expressed genes were identified from the raw data, which was reduced to 307 genes once duplicate genes were removed. Fully annotated microarray data have been deposited in BμG@ base (accession number E-BUGS-158) and also ArrayExpress (accession number E-BUGS-158).
3
Profiling Differential lncRNA and mRNA Expression
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LncRNA and mRNA expressions were determined using a CapitaBio Technology Human LncRNA Array v4 (CapitalBio, Beijing, China). Double‐stranded complementary DNAs (cDNAs) were synthesized from 1 μg total RNA. cDNA was labeled with Cy3‐dCTP or Cy5‐dCTP (GE Healthcare, Piscataway, NJ) and hybridized onto a human lncRNA + mRNA Array V4.0 (4 × 180 K; Agilent, Santa Clara, CA), comprising 40,916 human lncRNAs and approximately 34,000 human mRNAs. The lncRNA and mRNA target sequences were merged from ENSEMBL, human long intervening/intergenic ncRNA, and the LNCipedia catalog.
Threshold values of ≥2 and ≤2‐fold change and a Benjamini‐Hochberg corrected p = .05 were used to identify differentially expressed genes. The data were analyzed using hierarchical clustering with average linkage. Java Treeview software (Stanford University School of Medicine, Stanford, CA) was employed to visualize the microarray results. The primers are listed in Table S1.
Threshold values of ≥2 and ≤2‐fold change and a Benjamini‐Hochberg corrected p = .05 were used to identify differentially expressed genes. The data were analyzed using hierarchical clustering with average linkage. Java Treeview software (Stanford University School of Medicine, Stanford, CA) was employed to visualize the microarray results. The primers are listed in Table S1.
4
RNA Extraction and Microarray Analysis
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TRIzol® Reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA; cat. no. 15596-026); NucleoSpin® RNA clean-up (cat. no. 740.948.250); Nucleospin® Extract II (cat. no. 740.609.250; both Machery-Nagel, GmbH, Düren, Germany); GeeDom bio-chip universal label reagent (Capital Biotechnology, Co., Ltd., Beijing, China; cat. no. 360069); Cy3-dCTP (GE Healthcare, Chicago, IL, USA; cat. no. PA53031); Cy5-dCTP (GE Healthcare; cat. no. PA55031); 10% SDS; 20 × sodium citrate buffer (both Capital Biotechnology, Co., Ltd.); Agilent one-color RNA Spike-in kit (cat. no. 5188-5282); Agilent two-color RNA Spike-in kit (cat. no. 5188-5279; both Agilent Technologies Inc., Santa Clara, CA, USA); RNAiso Plus kit (Takara Biotechnology Co., Ltd., Dalian, China); TransScript One-step gDNA Removal and cDNA Synthesis SuperMix kits; TransStart Tip Green qPCR Super MixSYBR; Reverse Transcriptase kit; and TaqMan MGB probes (all Beijing Transgen Biotech Co., Ltd., Beijing, China) were purchased for use in the present study.
5
Fluorescence Microscopy Imaging Protocol
6
Fluorescent Labeling of AP-site Phagemids
7
Array CGH Analysis of DNA Samples
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Array CGH analysis was performed using standard methods described [5 (link)]. In brief, 300 ng of genomic DNA was labeled with Cy3-dCTP or Cy5-dCTP (GE Healthcare, Belgium) using Bioprime array CGH genomic labeling system (Invitrogen, Belgium). For the labeling, we used the “triangle method”: DNA samples from patients and controls were labeled and hybridized using a dye swap in trios consisting of at least one control per triangle. Samples were hybridized on 244K arrays (design ID 014693, Agilent, Belgium) for 40 h at 65°C. After washing, the samples were scanned at 5 μm resolution using a DNA microarray scanner G2505B (Agilent, Belgium). The scan images were analyzed using the feature extraction software 9.5.3.1 (Agilent) and further analyzed with “arrayCGHbase” [6 (link)]. Copy number variations were taken into consideration when two or more flanking probes were exceeding a value of the intensity ratios ± four times the standard deviation of log2 of all intensity ratios for that experiment. Always two experiments investigating the same sample with a dye swap were compared and only when an alteration is present in both experiments was the region included for further analysis. Inconsistencies were inspected manually.
8
Optimized EMSA Labelling and Binding Assay
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Oligonucleotides used for EMSA are listed in Supplementary Table 6 . For AP1, AP1m1, AP1m2, AGI-II and S-AGI, complementary single-stranded oligonucleotides were annealed in annealing buffer (10 mM Tris pH 7.5, 150 mM NaCl and 1 mM EDTA). The resulting double-stranded DNA with a protruding G was fluorescently labelled by end filling: 4 pmol of double-stranded DNA was incubated with 1 unit of Klenow fragment polymerase (Ozyme) and 8 pmol Cy3-dCTP or Cy5-dCTP (GE Healthcare) in Klenow buffer during 2 h at 37 °C, followed by 10 min enzyme inactivation at 65 °C. Binding reactions were performed in 20 μl binding buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% glycerol, 0.25 mM EDTA, 2 mM MgCl2, 0,01% Tween-20 and 3 mM TCEP) with 10 nM labelled probe, 1 × (28 ng ml−1) fish sperm DNA (Roche) as nonspecific competitor and 25–500 nM proteins.
Competition assays were performed in duplicates and 1–100 × fish sperm DNA (Roche) was used in the binding reaction. Signal quantification was performed in using ImageLab v2.0.1 (Bio-Rad Laboratories). Signal of each protein–DNA complex was quantified relatively to total DNA signal. ForFig. 4e , each binding reaction was performed in triplicate. Uncropped gels are presented as Supplementary Fig. 10 .
Competition assays were performed in duplicates and 1–100 × fish sperm DNA (Roche) was used in the binding reaction. Signal quantification was performed in using ImageLab v2.0.1 (Bio-Rad Laboratories). Signal of each protein–DNA complex was quantified relatively to total DNA signal. For
9
Genomic DNA Labeling and Analysis
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Four hundred nanograms of genomic DNA was labeled with Cy3-dCTP (GE Healthcare, Machelen, Belgium) using a Bioprime array CGH genomic labeling system (Thermo Fisher Scientific, Waltham, MA, USA). In parallel, Kreatech gender-matched controls were labeled with Cy5-dCTP. Samples were hybridized on the custom array CGH arrays for 40 h at 65 °C. After washing, the samples were scanned at 5 µm resolution using a DNA microarray scanner, G2505B (Agilent Technologies, Santa Clara, CA, USA). The scan images were analyzed using the feature extraction software 9.5.3.1 (Agilent Technologies). Segmentation was achieved using the circular binary segmentation algorithm in the DNACopy R package. Visual inspection and creation of the copy number profile plots were performed with ‘Vivar’ [29 (link)]. All of the raw array CGH data files were made publically available through the GEO website using the accession number GSE85444.
10
Porcine Genome Expression Analysis via NimbleGen Microarray
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The RNA samples were sent to KangChen Bio-tech, China, for microarray hybridization. Each RNA sample from different PAM treatments was hybridized to one Roche NimbleGen Porcine Genome Expression Array (Roche). Briefly, double-stranded cDNA was synthesized from 6 mg of total RNA using a T7-oligo (dT) primer. The cDNA was further purified and converted into cRNA using an in vitro transcription reaction. Five mg cRNA was reverse transcribed to cDNA, fragmented, and then labeled with Cy3-dCTP (GE Healthcare) using Klenow. These labeled cDNA fragments were hybridized to NimbleGen Porcine Genome Expression Arrays for 16 h at 42°C using the Roche NimbleGen Hybridization System. Afterwards, the GeneChips were washed, stained, and then scanned with a Roche-NimbleGen MS200. The Roche NimbleGen Porcine Genome Expression Array contains over 135,000 probe sets, representing 45,023 transcripts and variants of pig from the database of RefSeq, Unigene and TIGR.
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