A library of mouse TNFα DNAzymes (Table S2 ) predicted with a customized algorithm were screened in RAW264.7 cells. 200 nM of each DNAzyme was transfected into RAW264.7 cells using Oligofectamine according to manufacturer’s protocol. After 24 h incubation, the cell medium was collected for ELISA analysis of secreted TNFα. QIAzol was then added into the wells to lyse the cells and total RNA was isolated using RNeasy Mini Kit per manual. RNA was reverse transcribed using High-Capacity cDNA Reverse Transcription Kit. The TNFα mRNA level was quantified by qRT-PCR using PerfeCTa SYBR Green FastMix Reaction Mixes (QuantaBio) with 0.5 μM of custom-designed primers (Table S3 ) with Applied Biosystems StepOnePlus™ real time PCR system. The relative quantification of TNFα mRNA level was determined using ΔΔCt method with 18s mRNA as a reference.
Perfecta sybr green fastmix reaction mixes
Manufactured by Quantabio
PerfeCTa SYBR Green FastMix Reaction Mixes is a ready-to-use qPCR master mix that contains a proprietary, chemically-modified hot-start DNA polymerase, SYBR Green I dye, optimized buffer, and stabilizers. The product is designed for fast, sensitive, and reproducible quantitative PCR amplification.
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Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Miscript primer assay, by Qiagen (2 mentions)
Miscript 2 rt kit, by Qiagen (2 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000 platform, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Atac seq kit, by Diagenode (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Mini protean tbe urea gel, by Bio-Rad (1 mentions)
Quant it oligreen ssdna assay kit, by Thermo Fisher Scientific (1 mentions)
Mouse tnf α elisa kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Qscript cdna supermix, by Quantabio (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Nebnext ultra 2 q5 master mix, by New England Biolabs (1 mentions)
7 protocols using perfecta sybr green fastmix reaction mixes
1
Screening of Mouse TNFα DNAzymes
2
ATAC-seq Library Preparation and Analysis
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ATAC-seq libraries were prepared as previously described93 (link) using the ATAC-seq kit (Diagenode, C01080002) and 24 UDI for Tagmented libraries set II (Diagenode, C01011036). Briefly, 50,000 nuclei were pelleted and resuspended with tagmentase for 30 min at 37 °C. The tagmented DNA was then purified and used for library generation through PCR amplification. Size selection was conducted using AMPure XP beads (Beckman coulter, A63881), and the purified ATAC-seq libraries were sequenced on the Illumina NovaSeq 6000 platform.
For ATAC-qPCR experiments, the final elution product of the ATAC protocol was diluted 1:80. qPCR was conducted using PerfeCTa SYBR Green FastMix Reaction Mixes (QuantaBio, 95072-012) and a StepOnePlus™ Real-Time PCR System (Thermo Fischer Scientific, brand Applied Biosystems). Genomic DNA was utilized as a control for primer variability. All primer sequences used for qPCR are in Supplementary Data6 . The ATAC-qPCR results were first normalized by subtracting the Ct value of genomic DNA, yielding a normalized Ct value. The deltaCt value was then calculated by subtracting the normalized Ct value of a genomic locus near olfactory receptor gene (OR1A1), which exhibits very low ATAC-seq signal and low transcription levels in TSCs. The data was presented as negative deltaCt values, where an increase in value corresponds to increased openness.
For ATAC-qPCR experiments, the final elution product of the ATAC protocol was diluted 1:80. qPCR was conducted using PerfeCTa SYBR Green FastMix Reaction Mixes (QuantaBio, 95072-012) and a StepOnePlus™ Real-Time PCR System (Thermo Fischer Scientific, brand Applied Biosystems). Genomic DNA was utilized as a control for primer variability. All primer sequences used for qPCR are in Supplementary Data
3
DNAzyme-mediated Transcript Knockdown
4
Quantification of miR-33 Expression
5
Gene Expression Quantification by qPCR
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cDNA synthesis was performed from 500 ng of total RNA using qScript™ cDNA SuperMix (QuantaBio, 101414-108). The synthesized cDNA was diluted (x20), and 2 μL of the diluted cDNA was used for each reaction. qPCR was performed using PerfeCTa SYBR Green FastMix Reaction Mixes (QuantaBio, 95072-012) and a StepOnePlus™ Real-Time PCR System (Thermo Fischer Scientific, brand Applied Biosystems). Primers were designed to amplify the junction between two exons using a web-based primer design program, Primer3 (https://primer3.ut.ee/ ). All primer sequences used for qPCR are shown in Supplementary Data 6 . Relative transcript abundance was normalized to GAPDH as a loading control. Data were calculated as relative to control data using the 2−ΔΔCT method.
6
RNA Extraction and qRT-PCR Analysis
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Colo741 and FLX1 cells were seeded and treated in the same manner as described for immunoblotting in preparation for siRNA treatments. RNA was extracted with Trizol reagent (Thermo Fisher) according to the manufacturer’s instructions and quantified with a NanoDrop instrument. Normalized RNA was reverse transcribed with SuperScript II Reverse Transcriptase (Invitrogen). cDNAs were added to PerfeCTa SYBR Green FastMix Reaction Mixes (QuantaBio) and respective primers and analyzed using the BioRad CFX Connect Real-Time PCR Detection System. Primers were designed with Primer3 and obtained from Elim Biopharmaceuticals (Supplementary file 5 ). Quality control was performed for each primer using amplification and melting curves. All experiments were done in at least biological duplicate with three technical replicates per condition. If only one technical replicate did not show an appropriate amplification or melting curve, it was excluded from analyses.
7
Omni ATAC-seq Protocol for Nuclei
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Omni ATAC-seq was performed according to Corces et al 2017. Briefly, 50,000 cells were washed in ATAC-resuspension buffer (RSB) with 0.1% Tween-20 and then lysed by incubating on ice for 3 minutes in RSB with 0.1% NP-40 and 0.1% Tween-20. Lysis was washed out in RSB 0.1% Tween-20 and nuclei were pelleted by centrifugation at 600g/4°C/5 minutes. Nuclei were then incubated in the transposition mixture for 30 minutes at 37°C while shaking at 1000rpm. DNA was then purified using Zymo DNA clean and concentrator-5 Kit (cat# D4014). DNA was then amplified for 5 cycles with ATAC index primers and NEBNext Ultra II Q5 Master Mix (NEB #M0544). Additional amplification cycles were then determined by qPCR using 5uL of original amplification with PerfeCTa SYBR Green FastMix Reaction Mixes (Quantabio 95072–012). After additional amplification cycles with remaining 15uL DNA was then purified using Zymo DNA clean and concentrator-5 Kit (cat# D4014) and quantified by QUBIT.
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