Chromatin immunoprecipitation was performed according to the Upstate (Millipore) standard protocol. Briefly, cells were fixed using 1% formaldehyde for 10 min at 37°C, chromatin was extracted and sonicated to generate fragments between 200 and 500 bp. Next, 30 μg of sheared chromatin was immunoprecipitated overnight with the indicated antibody. Immunocomplexes were recovered using 20 μL of protein A magnetic beads, washed and eluted. Cross-linking was reversed at 65°C overnight and immunoprecipitated DNA was recovered using the IPure Kit (Diagenode). Genomic regions of interest were identified by real-time PCR (qPCR) using SYBR Green Master Mix (Invitrogen) and specific oligonucleotides in a QuantStudio™ 5 Real-Time PCR System machine (ThermoFisher Scientific). Each value was corrected by the corresponding input chromatin sample.
Quantstudio 5 real time pcr system machine
Manufactured by Thermo Fisher Scientific
The QuantStudio 5 Real-Time PCR System is a laboratory instrument used for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It is designed to accurately and reliably measure gene expression levels, detect and quantify nucleic acid sequences, and perform a variety of other real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green pcr master mix, by Thermo Fisher Scientific (6 mentions)
Upstate, by Merck Group (2 mentions)
Ipure kit, by Diagenode (2 mentions)
Sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Maxima reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Picopure rna isolation system, by Thermo Fisher Scientific (2 mentions)
Molecular grade water, by Thermo Fisher Scientific (2 mentions)
Random primer, by Promega (2 mentions)
Genelute mammalian total rna miniprep kit, by Merck Group (2 mentions)
On column dnase 1 digestion set, by Merck Group (2 mentions)
Kapa sybr fast qpcr master mix, by Roche (2 mentions)
Superscript 2, by Thermo Fisher Scientific (2 mentions)
Rnaseout, by Thermo Fisher Scientific (2 mentions)
Quantstudio 12k flex, by Thermo Fisher Scientific (2 mentions)
Dntps, by Promega (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Cfx opus 384 real time pcr system, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
7 protocols using quantstudio 5 real time pcr system machine
1
ChIP-qPCR for Transcription Factor Binding
2
ChIP-qPCR for Transcription Factor Binding
3
Quantitative Analysis of Testis mRNA
4
Testis mRNA Extraction and qRT-PCR Analysis
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Whole testes (n=20-25) were isolated and mRNA was extracted with PicoPure RNA Isolation system (Applied Biosystems) following the manufacturer’s instructions. Reverse transcription was performed using Maxima Reverse Transcriptase (ThermoFisher) as per manufacturer’s instructions and using 0.5μg of RNA as template. qRT-PCR was performed using SYBR Green PCR Master Mix (ThermoFisher) and a QuantStudio 5 Real-Time PCR System machine (Applied Biosystems). 8 replicates were performed for the experiments in Figures 5 H and 4 replicates for each experiment shown in Figure S3 E. To detect Fsisoform B (FlyBase code FBtr0339996), we used the following primers and normalized expression levels to the control gene α-tub84B:
Fs-fwd: 5′-AGTGTCATATATACTCTCCGCATGT-3′
Fs-rev: 5′-ACAGCAACTGCTTTTTAACTATGCC-3′
α-tub84B-fwd: 5′-TCGTTTTACGTTTGTCAAGCCTC-3′
α-tub84B-rev: 5′-GAGATACATTCACGCATATTGAGTT-3′
daw-fwd: 5′-CCCATCTTCGACGGGATGAC-3′
daw-rev: 5′-TTGCACTCGACCTCCTCTCT-3′
We failed to detect transcripts of Fs isoform A in the testis (FlyBase FBtr0087398), using the following primers and normalized expression levels to the control gene α-tub84B:
Fs-A-fwd:5′-GAACGGACCGCGCTAAAAAT-3′
Fs-A-rev:5′-GGCAAACGCACTGGTTTCAT-3′
Fs-fwd: 5′-AGTGTCATATATACTCTCCGCATGT-3′
Fs-rev: 5′-ACAGCAACTGCTTTTTAACTATGCC-3′
α-tub84B-fwd: 5′-TCGTTTTACGTTTGTCAAGCCTC-3′
α-tub84B-rev: 5′-GAGATACATTCACGCATATTGAGTT-3′
daw-fwd: 5′-CCCATCTTCGACGGGATGAC-3′
daw-rev: 5′-TTGCACTCGACCTCCTCTCT-3′
We failed to detect transcripts of Fs isoform A in the testis (FlyBase FBtr0087398), using the following primers and normalized expression levels to the control gene α-tub84B:
Fs-A-fwd:5′-GAACGGACCGCGCTAAAAAT-3′
Fs-A-rev:5′-GGCAAACGCACTGGTTTCAT-3′
5
Comparative COVID-19 RT-PCR Detection
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The nasopharyngeal swabs collected from participants were transported using viral transport medium (VTM). All methods were carried out in accordance with relevant guidelines and regulations. In the lab, RNA extraction was performed by the automated machine MGISP-960, as per the manufacturer's instructions. After the RNA extraction, 10 microliters of the sample extract was added to 20 and 15 microliters of the master mix for the BGI Genomics' 2019-nCoV Fluorescence Detection Real-Time RT-PCR kit and the Thermo Fisher Applied Biosystems TaqPath COVID-19 CE-IVD RT-PCR kit, respectively. Both the extraction and the PCR detection methods were verified in house. The real time fluorescent RT-PCR was performed using the Bioer LineGene 9600 Plus Fluorescent Quantitative Detection System for BGI Genomics' 2019-nCoV Fluorescence kit and on the QuantStudio 5 Real-Time PCR System machine for the Applied Biosystems TaqPath kit.
6
RNA Extraction and qPCR Analysis
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Total RNA was extracted from cells using GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) and On-Column DNase I Digestion set (Sigma-Aldrich), or using RNeasy Mini kit and RNase-free DNase (Qiagen). Complementary DNA was synthesized from the RNA using random primers (Promega), dNTPs (Promega), RNAseOUT (Invitrogen), and SuperScript II (Invitrogen), or using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time PCR was performed using KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) on QuantStudio 12 K Flex and QuantStudio 5 Real-Time PCR System machines (Thermo Fisher Scientific) or using SYBR Green PCR Master Mix (Thermo Fisher Scientific) on CFX Opus 384 Real-Time PCR System (Bio-Rad). Molecular grade water (Thermo Fisher Scientific) was used when necessary. Each gene expression level was normalized by the average expression level of PBGD. Primer sequences are shown in table S1.
7
Total RNA Extraction, cDNA Synthesis, and qPCR Analysis
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Total RNA was extracted from cells using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) and the On-Column DNase I Digestion set (Sigma-Aldrich). Complementary DNA was synthesized from the RNA using random primers (Promega), dNTPs (Promega), RNAseOUT (Invitrogen) and SuperScript II (Invitrogen). Real-time PCR was performed with KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) on QuantStudio 12K Flex and QuantStudio 5 Real-Time PCR System machines (Thermo Fisher Scientific). Molecular grade water (Thermo Fisher Scientific) was used when necessary. Each gene expression level was normalized by the average expression level of PBGD. Primer sequences are shown in S1 Table .
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