For DNA microarray hybridization, RNA was pooled by mixing equal amounts of total RNA, and biotin-labeled cRNA targets were synthesized starting from 1.5 μg of total RNA. Double-stranded cDNA synthesis was performed using the Illumina® TotalPrep RNA Amplification Kit (Illumina, San Diego, CA, USA), while biotin-UTP-labeled antisense RNA was transcribed in vitro using the Ambion MEGAscript kit (Ambion Life Technologies, Carlsbad, CA, USA). All steps of the labeling procedure were performed according to the manufacturers' protocols. Microarray experiments were conducted on the HumanHT-12 v4 Sentrix Expression BeadChip (Illumina), which contains 47,231 probes, representing 31,332 annotated genes. Hybridization of labeled cRNA to the BeadChip, washing, and scanning were performed according to the Illumina Bead Station 500× manual.
Ambion megascript kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ambion MEGAscript kit is a high-yield in vitro transcription kit designed for the production of large amounts of RNA from DNA templates. The kit includes the necessary reagents and enzymes to efficiently generate high-quality RNA from a DNA template.
Automatically generated - may contain errors
Lab products found in correlation
Totalprep rna amplification kit, by Illumina (3 mentions)
Beadstation 500, by Illumina (2 mentions)
Humanht 12 v4 sentrix expression beadchip, by Illumina (2 mentions)
Genomestudio v2011, by Illumina (1 mentions)
Rna isolation kit, by GE Healthcare (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Phosphate buffer, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Sartorius (1 mentions)
Pbluescript 2 sk, by Agilent Technologies (1 mentions)
Lr clonase, by Thermo Fisher Scientific (1 mentions)
Gateway system, by Thermo Fisher Scientific (1 mentions)
Pentr topo cloning vector, by Thermo Fisher Scientific (1 mentions)
9 protocols using ambion megascript kit
1
Microarray Expression Profiling Protocol
2
Transcriptomic Profiling of Total RNA
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Total RNA was extracted using an RNA Isolation kit (GE Healthcare Life Sciences, Logan, UT, USA) and then pooled for DNA microarray hybridization. Biotin‐labeled cRNA targets were synthesized using 1.5 μg of total RNA, and an Illumina® TotalPrep RNA Amplification kit (Illumina, San Diego, CA, USA) was used to synthesize double‐stranded cDNA. Biotin‐UTP‐labeled antisense RNA was transcribed in vitro using an Ambion MEGAscript kit (Ambion Life Technologies, Carlsbad, CA, USA). All labeling procedures were performed according to the manufacturers' protocols. Microarray experiments were conducted using HumanHT‐12 v4 Sentrix Expression BeadChips (Illumina). Hybridization of labeled cRNA to the BeadChip, washing and scanning were performed according to the Illumina Bead Station 500× instructions. Raw data were extracted using the software provided by the manufacturer (Illumina GenomeStudio v2011.1 [Gene Expression Module v1.9.0]). The local‐pooled‐error (LPE) test was used to determine the statistical significance of differences in expression data, and the false‐discovery rate was controlled for given p‐values using the Benjamini–Hochberg algorithm. Microarray data are deposited in Gene Expression Omnibus (GEO) with the accession number: GSE89673.
The private accession link:
(https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=qbypgquqtfsxnyb&acc=GSE89673 )
The private accession link:
(
3
Quantitative Analysis of Gene Expression
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Paraformaldehyde, sucrose, phosphate buffer, EDTA, and proteinase K were from Sigma-Aldrich. Superfrost/Plus slides were from Thermo Fisher Scientific. Slide mailers were from Baxter Scientific. Primers and probes for real-time polymerase chain reaction (RT-PCR) used to quantitate expression levels of Klotho, Fgfr1c, Atp1a1, and Slc12a2 were from Applied Biosystems. Supplementary Table S1 lists the ABS assays used [20 (link)]. The probes for in situ hybridization were prepared using the Ambion MEGAscript kit, and digoxigenin-11-UTP from Boehringer Mannheim, following the manufacturer's instructions.
4
RNA Extraction and Microarray Hybridization
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For DNA microarray hybridization, RNA was extracted from HUVECs using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA was pooled by mixing equal amounts of total RNA, and biotin-labeled cRNA targets were synthesized from 1.5 μg of total RNA. Double-stranded cDNA synthesis was performed using the Illumina® TotalPrep RNA Amplification Kit (Illumina, Inc., San Diego, CA, USA), while biotin-UTP-labeled antisense RNA was transcribed in vitro using the Ambion MEGAscript kit (Ambion Life Technologies, Carlsbad, CA, USA). All steps of the labeling procedure were performed according to the manufacturers’ protocols. Microarray experiments were conducted on the HumanHT-12 v4 Sentrix Expression BeadChip (Illumina) containing 47,231 probes representing 31,332 annotated genes.
5
Generating Pseudotyped HCV Virus Particles for Infection Assays
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The following plasmids were provided by Dr. Takaji Wakita at Tokyo Metropolitan Institute of Neuroscience: Genotype 2a HCV genomic RNA clone pJFH1 (APP1025) (38 (link)). pNL4.3.Luc_RΔenv provided by Dr. N.R. Landau (39 (link)), pHCV_H77_E1_E2(AF009606) Dr. Joe Grove (Addgene) (40 ). For single-round infection assay, human embryonic kidney 293T/17 cells (ATCC, CRL-11268) were co-transfected with pNL4.3.Luc_RΔenv, containing firefly luciferase gene at the nef position (1.35 ug) and genotype 1a pHCV_H77_E1_E2(AF009606) (0.6 ug). Transfection was performed in 293T/17 cells using genejuice (Novagen, USA) transfection kit according to manufacturer's protocol. At day 3 or day 4, pseudotyped HCV virus particles were harvested and filtered over 0.45 um nitrocellulose membrane (SartoriusStedim, Gottingen, Germany). Replicative JFH1-AM120-Rluc in vitro transcribed RNA, containing a luciferase reporter gene, was generated according to manufacturer's instructions (Ambion MEGAscript-kit, ThermoFisher, USA) and electroporated into Huh 7.5 cells as previously described (41 (link)). Virus particles were harvested on day 8 and, TCID50s were determined. The TCID50 of HCV ranged from 2 × 103 to 4 × 103.
6
Quantification of rRNA Copies via Standard Curve
7
Gateway Cloning of Bub1 and AP-1-2-beta
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pAGW-Bub1 and pAFW-AP-1-2-beta were constructed with the Gateway system (Invitrogen). Bub1 and ap-1-2-beta full-length cDNAs were amplified from LD22858 and w1118 genomic cDNAs, respectively, by using the primers listed in Table S1 in the supplemental material and then subcloned into the pENTR TOPO cloning vector (Invitrogen). These pENTR vectors were subsequently recombined into FLAG- or GFP-tagged destination vectors (Carnegie Institute for Science) by using LR Clonase (Invitrogen).
dsRNA was constructed by using an Ambion MEGAscript kit (Thermo) and stored at −20°C.
The sequences used are listed in Table S1 in the supplemental material.
dsRNA was constructed by using an Ambion MEGAscript kit (Thermo) and stored at −20°C.
The sequences used are listed in Table S1 in the supplemental material.
8
Synthesis of Double-Stranded RNA
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Double-stranded RNAs were prepared as described in Amukamara et al., 2020 (link). Briefly, DNA templates were prepared by PCR using gene-specific primers (Amukamara et al., 2020 (link)). Sense and anti-sense RNA were transcribed together with an Ambion MEGAscript kit (ThermoFisher Sci, Waltham, MA) and allowed to anneal to form a 404 bp ds-Dnmt1 RNA. The concentration of dsRNA was adjusted to 3 μg/μL in injection buffer (5 mM KCl, 0.1 mM NaH2PO4).
9
Synthesis of Double-Stranded Dnmt1 RNA
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Double-stranded RNAs were prepared as described in Amukamara et al. 2020 . Briefly, DNA templates were prepared by PCR using gene-specific primers (Amukamara et al. 2020) . Sense and anti-sense RNA were transcribed together with an Ambion MEGAscript kit (ThermoFisher Sci, Waltham, MA) and allowed to anneal to form a 404 bp ds-Dnmt1 RNA. The concentration of dsRNA was adjusted to 3 µg/µL in injection buffer (5 mM KCl, 0.1 mM NaH2PO4).
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