Total RNAs were extracted from skin tissues, HEM or B16F0 cells using NucleoZOL kit (Macherey-Nagel, 740404). Complementary DNA (cDNA) was prepared from 1 ug of total RNAs using a reverse transcription kit (Intron, 25087) and subjected to PCR analysis to determine the levels of each transcript, such as POMC, MITF-M, CREB, CRTC1, β-catenin, TYR, TRP-1, DCT, PMEL17, Rab27A or β-catenin. Nucleotide sequences of PCR primers were described in Table S1 . RT-PCR condition was as follows: reverse transcription at 95°C for 3 min followed by 25-30 cycles of PCR at 94°C for 30 sec (denaturation), 50-60°C for 1 min (annealing), and 72°C for 1 min (extension). Amplified transcripts were resolved on agarose gels by electrophoresis and visualized by staining with EcoDye (Biofact, ES301).
Nucleozol kit
Manufactured by Macherey-Nagel
Sourced in Germany
The NucleoZOL kit is a product offered by Macherey-Nagel, a leading provider of laboratory equipment. The kit is designed for the isolation and purification of RNA from a variety of biological samples. It utilizes a guanidinium thiocyanate-phenol-based extraction method to efficiently extract high-quality RNA.
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Lab products found in correlation
Ecodye, by Biofact (2 mentions)
Hiscript 2 q rt supermix reagent kit, by Vazyme (2 mentions)
Kod hot start dna polymerase, by Merck Group (1 mentions)
Nanojuice transfection kit, by Merck Group (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (1 mentions)
Hiscript 3 rt supermix for qpcr, by Vazyme (1 mentions)
Ribo zero plant, by Illumina (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Nanodrop one microvolume uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Sybr green pcr master mix, by Vazyme (1 mentions)
10 protocols using nucleozol kit
1
Skin Tissue RNA Extraction and RT-PCR Analysis
2
Constructing Lamin-GFP and Gelsolin-GFP-KASH2 Fusion Proteins
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Plasmids expressing lamin A and lamin B1 fused to GFP were a kind gift from Tom Misteli [38 (link)] and pEGFP-KASH2 and pEGFP-KASH2ext were a kind gift from Didier Hodzic [39 (link)]. Gelsolin was cloned by RT-PCR: for reverse transcription, total RNA was isolated from B16–F10 by the NucleoZOL kit (740404.200, MACHEREY-NAGEL, Düren, Germany) according to the manufacturer’s instructions and reverse-transcribed using GoScript™ Reverse Transcription System (Promega) and the oligonucleotide 5′-GAGAACTGAAACCTGGGT-3′. The N-terminal part of Gelsolin (aa 1–370) was amplified by PCR using KOD Hot Start DNA Polymerase (71086, Merck, Kenilworth, NJ, USA) and the oligonucleotides 5′-cagctagccaccATGGTGGTGGAGCACCCC-3′ and 5′-gcaccggtaTGGGGTACTGCATCTTGGAGAT-3′. The PCR product was ligated into NheI-AgeI sites in pEGFP-KASH2ext upstream to GFP to generate pGSN-EGFP-KASH2ext. Transfection of cells was carried out by the Nanojuice transfection kit (71900-3, Merck, Kenilworth, NJ, USA) following the manufacturer’s instructions.
3
Quantitative Analysis of Melanogenic Regulators
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Total RNAs were prepared with NucleoZOL kit (Macherey-Nagel, 1609), and subjected to RT-PCR analysis in the determination of mRNA levels of MITF-M, TYR, β-catenin, SOX10, KPNA subtype or CRTC1 with the internal control β-actin. Nucleotide sequences of RT-PCR primers are described in Table S1 . Briefly, total RNAs were reversely transcribed for 1 h at 42℃ with oligo-dT as a primer (iNtRON, 25087). Single-stranded cDNAs were subjected to 27-40 cycles of PCR using a premix kit (Bioneer, K2018), in which one cycle consisted of denaturation for 15-30 s at 95℃, annealing for 15-30 s at 54-60℃, and DNA extension for 25-60 s at 72℃. RT-PCR products were resolved on agarose gels by electrophoresis and stained with EcoDye (Biofact, ES301-1000).
4
Quantitative RT-PCR Analysis of DDX56 Expression
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In brief, we isolated total tissue and cellular RNAs using the NucleoZOL Kit (Macherey-Nagel, Düren, Germany). Subsequently, total RNA (1000 ng) was prepared into cDNA with the HiScript III RT SuperMix for qPCR (R323-01, Vazyme, Nanjing, China) through reverse transcription, followed by preservation under −80°C. Using ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme), this study then conducted quantitative RT-PCR with the 7500 Real-time PCR system (ThermoFisher, USA). The 2-ΔΔCt method was applied in calculating relative gene levels as fold-change (FC), followed by normalization based on GAPDH expression. In quantitative QRT-PCR, the primers used were shown below: DDX56, 5’-CCGCTTATGCTATTCCGATGC-3’ (F), 5’-GCTCCTTGGTAGGAACAAGAACA-3’ (R); GAPDH, 5’-GGAGCGAGATCCCTCCAAAAT-3’ (F), 5’-GGCTGTTGTCATACTTCTCATGG-3’ (R). The normalized value of mRNA level was set to 1 for cells transfected with control plasmid.
5
Evaluating Osteogenic Potential of Nanostructured Scaffolds
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To evaluate the osteogenic properties of nanostructured scaffolds incorporated with HA, ATP, and both combined, BMSCs were seeded on nanostructured scaffolds in a 6-well plate. At each timepoint (1, 3, and 7 days), total RNA was extracted using a NucleoZol kit (Macherey-Nagel, Germany) according to the instructions. After that, reverse transcription was carried out to synthesize cDNA using the HiScript II Q RT SuperMix reagent Kit (Vazyme, China). Then, gene amplification was performed using the SYBR Premix Ex Taq-TM Real-time PCR kit (Vazyme, China), and the osteogenesis-related primers (alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), Osterix (OSX), and Runt-related factor 2 (Runx2) used in this study are listed in Table 1 .
Primer Sequences Applied to RT–PCR Analysis
| Gene | Forward Primer | Reverse Primer |
|---|---|---|
| 5’-CACCACCAACTGCTTAGC-3’ | 5’-TTCACCACCTTCTTGATGTC-3’ | |
| 5’-GCAGTATGAATTGAATCGGAACAAC-3’ | 5’-ATGGCCTGGTCCATCTCCAC-3’ | |
| 5’-TACGACCATGAGATTGGCAGTGA-3’ | 5’-TATAGGATCTGGGTGCAGGCTGTAA-3’ | |
| 5’-CTGCAAGCAGTATTTACAACAGAGG-3’ | 5’-GGCTCACGTCGCTCATCTT-3’ | |
| 5’-AGGCCTTTGCCAGTGCCTA-3’ | 5’-GCCAGATGGAAGCTGTGAAGA-3’ |
6
A. thaliana Leaf RNA-seq Library Prep
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Total RNA was extracted from leaves using the NucleoZOL kit (Macherey-Nagel, Hœrdt, France) followed by a purification with the Agencourt RNACleanup XP beads (Beckmann-Coulter, Villepinte, France). The sequencing libraries were constructed using the TruSeq Stranded Total RNA with Ribo-Zero Plant (Illumina Inc., San Diego, CA, USA) and then sequenced with a Nextseq500 in single-end 75 bp. The RNA-seq data was analysed as described [57 ] except for the following modifications. The reads were mapped with STAR v2.7.0c [58 (link)] on the A. thaliana genome available from the release 39 of EnsemblGenomes but the mitochondrial genome was replaced by the Col-0 mitochondrial genome from the NCBI under the accession BK010421. The mapping parameters were --outSAMprimaryFlag AllBestScore --outFilterMultimapScoreRange 0--alignIntronMax 1.
7
RNA Extraction and qRT-PCR Analysis
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Cell monolayers were washed twice with PBS and the total RNA was extracted using the NucleoZOL kit (Macherey-Nagel). The RNA pellets were resuspended in 20 uL of nuclease-free water. RNA quantification was achieved using the Nanodrop™ One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific). cDNA was generated using the iScriptTM cDNA Synthesis Kit (Bio-Rad) using 1μg of RNA. Amplification was achieved using the QuantiTect SYBR Green PCR Kit (Qiagen) in conjunction with Applied Biosystems™ 7500 Fast Real-Time PCR System analysis. Gene expression was determined relative to rPlp0 using the delta delta Ct method [2^(ΔΔCt)]. Primers were acquired from IDT (Integrated DNA Technologies), and primer optimization was determined using the standard curve method. Primer sequences are shown in Supplemental Table 1
8
Total RNA Extraction and Sequencing
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Total RNA was purified by the NucleoZOL kit (MACHEREY-NAGEL, 740404.200, Duren, Germany) according to the manufacturer’s instructions. Purified RNA was sent for poly A containing mRNA selection, library preparation, and sequencing at the Technion Genome Center. Replicates number was five for untreated control and migrating cells and three for cells treated with GSK343 or DRB.
9
Generating M1 Macrophages from RAW 264.7 Cells
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To produce M1 macrophages, RAW 264.7 cells were seeded onto six-well plates. When the cell density reached 70%–90%, the original media was changed to one containing 200 ng/mL lipopolysaccharide (LPS), and the culture was continued for an additional 8 h. Then, a portion of the cell samples were extracted and Quantitative Real-time Polymerase Chain Reaction (qPCR) analysis was performed according to the manufacturer’s protocol to determine M1-related genes, including those encoding IL-1, IL-6, and NOS2, to confirm the M1 phenotype. mRNA was collected using the NucleoZol kit (Machery-Nagel, Germany). Reverse transcription PCR was performed using the Hiscript II Q RT SuperMix reagent kit (Vazyme, China). SYBR Green PCR Master Mix (Vazyme, China) was employed using qPCR. The 2−ΔΔCt method was used to measure the expression of the target gene. the qPCR reactions were conducted in triplicate for both the target and the housekeeping gene GAPDH. Using a cell scraper, the remaining cells were removed before the scaffold and 2 × 105 cells per well were seeded in 6-well plates. At 1 and 3 days, cell samples from each group were collected for qPCR analysis. The primer sequences are shown in Supplemental Table S1 .
10
OVCAR8 Cell Transcriptomic Profiling
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OVCAR8 cells were cultured in CTL medium or ACM for 18 hours or cultured in CTL medium or ACM. The wells were washed with sterile PBS and the RNA was extracted (NucleoZOL kit; Macherey-Nagel). Tidyverse, EdgeR, and Heatmaply were used for bioinformatics analysis and heatmap generation.
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