ChIP assays were performed with HCT116 cells in accordance with standard protocols as described previously [35 (link)]. Normal rabbit IgG served as the control. ChIP samples were analyzed by quantitative real-time PCR using the FastStart Universal SYBR Green Master (Vazyme Biotech, China). The primer sequences for ChIP are listed in Additional file 2 : Table S6.
Faststart universal sybr green master
Manufactured by Vazyme
Sourced in China
FastStart Universal SYBR Green Master is a ready-to-use master mix for real-time PCR. It contains FastStart Taq DNA Polymerase, SYBR Green I, dNTPs, and optimized buffer components.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Transscript rt reagent kit, by Transgene (1 mentions)
Geneamp pcr system 9600, by PerkinElmer (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Biospectrometer, by Eppendorf (1 mentions)
Primescript rt reagent, by Takara Bio (1 mentions)
Steponeplus real time pcr system, by Vazyme (1 mentions)
Hiscript 1st strand cdna synthesis kit, by Vazyme (1 mentions)
Hiscript first strand cdna synthesis kit, by Vazyme (1 mentions)
Ab6002, by Merck Group (1 mentions)
Ab191250, by Abcam (1 mentions)
Simplechip plus sonication chromatin ip kit, by Cell Signaling Technology (1 mentions)
Rna pol 2, by Merck Group (1 mentions)
H3k4me3, by Abcam (1 mentions)
Stepone, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Vazyme (1 mentions)
7 protocols using faststart universal sybr green master
1
ChIP Assay with HCT116 Cells
2
Comprehensive Transcriptional Analysis of Cellular Metabolism
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Total RNA was extracted from cells using RNAiso Plus (TaKaRa, Japan, 108‐95‐2) and isolated according to the manufacturer's instructions. The RNA concentration and purity were measured by a BioSpectrometer (Eppendorf, Germany); 2 μg total RNA was reversely transcribed into cDNA using the TransScript RT reagent Kit (TransGen, China, AE301). According to the manufacturer's instructions, qRT‐PCR was performed with FastStart Universal SYBR Green Master (Vazyme, China, Q111) using a Gene Amp 9600 PCR system (Perkin‐Elmer, Waltham, MA). The relative amount of cDNA was analyzed using the 2−ΔΔCT method. The primers for qRT‐PCR used in this study were as follows: PDHA1‐Forward: CTTACCGCTACCATGGACACAGCATG,
Reverse: CTCCTTTAATTCTTCAACACTTGCAAGA; HK2‐Forward: GAGCCACCACTCACCCTACT, Reverse: CCAGGCATTCGGCAATGTG; PFK2‐Forward: ATTGCGGTTTTCGATGCCAC, Reverse: GCCACAACTGTAGGGTCGT; IDH1‐ Forward: TTGGCTGCTTGCATTAAAGGTT, Reverse: GTTTGGCCTGAGCTAGTTTGA; CS‐Forward: GAGCAGGCCAGAGTTAAGAC, Reverse: AAAATAAGCCCTCAGGTAGG; LDHA‐Forward: AAACGCGCCTTAATTTAGTCCA, Reverse:CAGCCGCTTCCAATAATACGG; PGC1α‐Forward: GTAAATCTGCGGGATGATGG, Reverse: AGCAGGGTCAAAATCGTCTG; SIRT1‐Forward: TGCCATCATGAAGCCAGAGA, Reverse: AACATCGCAGTCTCCAAGGA; and GAPDH‐Forward:CAAGAAGGTGGTGAAGCAGG, Reverse: CCACCCTGTTGCTGTAGCC.
Reverse: CTCCTTTAATTCTTCAACACTTGCAAGA; HK2‐Forward: GAGCCACCACTCACCCTACT, Reverse: CCAGGCATTCGGCAATGTG; PFK2‐Forward: ATTGCGGTTTTCGATGCCAC, Reverse: GCCACAACTGTAGGGTCGT; IDH1‐ Forward: TTGGCTGCTTGCATTAAAGGTT, Reverse: GTTTGGCCTGAGCTAGTTTGA; CS‐Forward: GAGCAGGCCAGAGTTAAGAC, Reverse: AAAATAAGCCCTCAGGTAGG; LDHA‐Forward: AAACGCGCCTTAATTTAGTCCA, Reverse:CAGCCGCTTCCAATAATACGG; PGC1α‐Forward: GTAAATCTGCGGGATGATGG, Reverse: AGCAGGGTCAAAATCGTCTG; SIRT1‐Forward: TGCCATCATGAAGCCAGAGA, Reverse: AACATCGCAGTCTCCAAGGA; and GAPDH‐Forward:CAAGAAGGTGGTGAAGCAGG, Reverse: CCACCCTGTTGCTGTAGCC.
3
Quantitative RT-PCR Analysis of DACT1 in Gastric Cancer
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DACT1 mRNA expression levels were analyzed by quantitative reverse transcription–polymerase chain reaction (PCR) using total RNA extracted from 76 pairs of cancerous and normal gastric tissue samples using Trizol reagent (Thermo Fisher Scientific). Total RNA was reversely transcribed to first-strand complementary DNA using Primescript RT Reagent (Takara, Otsu, Japan). The real-time PCR primers for DACT1 were as follows: forward primer 5′-TGTGAATCCCAAGTACCAGTGT-3′ and reverse primer 5′-CCGTCAGACAAAGGAGAAACATT-3′. β-Actin was used to normalize DACT1 gene expression levels and amplified with forward primer 5′-AGAAAATCTGGCACCACACC-3′ and reverse primer 5′-TAGCACAGCCTGGATAGCAA-3′. Amplification reactions were executed in a 10 µL reaction volume containing 0.2 µL primers, 5 µL Master mix, and 100 ng complementary DNA. The cycling conditions were set at 95°C for 5 minutes, followed by 40 cycles at 95°C for 10 seconds and 60°C for 30 seconds. Real-time PCR was carried out using FastStart Universal SYBR-Green Master (Vazyme, Nanjing, People’s Republic of China) with the StepOnePlus Real-Time PCR System in triplicate. The 2−ΔCT algorithm was used for calculating the expression of individual DACT1 relative to expression of β-actin.4 (link)
4
Quantitative RT-PCR Protocol for Gene Expression
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Total RNA from cultured cells was extracted using TRIzol reagent (Invitrogen). cDNAs were synthesized with a HiScript 1st Strand cDNA Synthesis Kit (Vazyme Biotech, China). Quantitative RT-PCR was performed using a FastStart Universal SYBR Green Master (Vazyme Biotech, China) according to the manufacturer’s instructions in a StepOnePlus™ Real-Time PCR System (Thermo Scientific) in a final volume of 20 μl. Cycling conditions were 94 °C for 15 s, 60 °C for 1 min, and 72 °C for 30 s. Each reaction was performed in triplicate. The primer sequences for RT-PCR are listed in Additional file 2 : Table S5.
5
Quantifying Tumor Suppressor Gene Expression
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Total RNA from tissues and cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNAs were synthesized with a HiScript first Strand cDNA Synthesis kit (VAzyme Biotech, Nanjing, P.R. China). The FastStart Universal SYBR Green Master (Vazyme Biotech) was used for quantitative real-time PCR (qRT-PCR) according to the manufacturer’s instructions to measure the expression level of certain genes. Primers: p16, 5′-ATGGAGCCTTCGGCTGACT-3′ (forward), 5′-GTAACTATTCGGTGCGTTGGG-3′ (reverse), p21, 5′-TGTCCGTCAGAACCCATGC-3′ (forward), 5′-AAAGTCGAAGTTCCATCGCTC-3′ (reverse), p27, 5′-TAATTGGGGCTCCGGCTAACT-3′ (forward), 5′-TGCAGGTCGCTTCCTTATTCC-3′ (reverse), PTEN, 5′-TTTGAAGACCATAACCCACCAC-3′ (forward), 5′-ATTACACCAGTTCGTCCCTTTC-3′ (reverse), p57, 5′-GCGGCGATCAAGAAGCTGT-3′ (forward), 5′-GCTTGGCGAAGAAATCGGAGA-3′ (reverse), p53 5′-GAGGTTGGCTCTGACTGTA CC-3′ (forward), 5′-TCCGTCCCAGTAGATTACCAC-3′ (reverse).
6
ChIP-qPCR Analysis of PTEN
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ChIP assays were performed with SUNE-1 cells using SimpleChIP® Plus Sonication Chromatin IP kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions. Normal rabbit IgG served as the control. ChIP samples were analyzed by qRT-PCR using the FastStart Universal SYBR Green Master (Vazyme Biotech). The percentage of ChIP DNA was calculated relative to the input DNA from primer-specific standard curves. The primer sequences of PTEN promoter for ChIP are 5′-GGAGGCAGCCGTTCGGAGGATTATT-3′ (forward), 5′-GGAAATGGCTCTGGACTTGGCGGTA-3′ (reverse) and 3′UTR region primer sequences are 5′-TCCTGGATGACCTTTGACATAC-3′ (forward), 5′-TCCAGTATGCCAACTTTGGTT-3′ (reverse). Antibodies used were: EZH2 (Abcam, ab191250), H3K27me3 (Abcam, ab6002), RNA polymerase II (RNA polII) (Millipore, 05-623), and H3K4me3 (Abcam, ab8580).
7
Transcriptome Analysis of Cell Samples
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Total RNA from cultured cells in cell culture plate, hydrogel, and mouse skin samples were extracted using Trizol reagent (Vazyme Biotech, China). QRT-PCR was performed using a FastStart Universal SYBR Green Master (Vazyme Biotech, China) in a StepOne (Thermo Fisher). The primer sequences for qRT-PCR were listed in Supplementary Table 1 .RNA-seq analysis was conducted by Novogene Co., LTD (Beijing, China).
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