Total RNA from mice tumors was extracted using an RNeasy Plus Mini Kit (Qiagen, Germantown, MD) and reverse transcription was conducted using a PrimeScript First Strand cDNA Synthesis Kit (Takara, Mountain View, CA). qPCR analysis was performed using SYBR Premix Ex Taq (Tli RNase H Plus) (Takara) and a CFX384 Touch instrument (Bio-Rad, Hercules, CA, USA). The primers used in this study: IFN-γ, 5′- GAAAGCCTAGAAAGTCTGAATAACT-3′ and 5′- ATCAGCAGCGACTCCTTTTCCGCTT-3′; IL-2, 5′-ATGTACAGCATGCAGCTCGCATC-3′ and 5′-GGCTTGTTGAGATGATGCTTTGACA-3′; IL-10, 5′-TGAAGACCCTCAGGATGCGG-3′ and 5′-AGAGCTCTGTCTAGGTCCTGG-3′; and β-actin, 5′- CGTGCGTGACATCAAAGAGAA-3′ and 5′-TGGATGCCACAGGATTCCAT-3′. PCR conditions were as follows. For IFN-γ, IL-2, and β-actin: initial denaturation at 95 °C for 1 min; 35 cycles of denaturation (95 °C for 30 s), annealing (60 °C for 30 s), and extension (72 °C for 1 min), and a final extension at 72 °C for 1 min. For IL-10: initial denaturation at 95 °C for 1 min, 40 cycles of denaturation (95 °C for 30 s), annealing (60 °C for 30 s), and extension (72 °C for 1 min); and a final extension at 72 °C for 1 min.
Cfx384 touch instrument
Manufactured by Bio-Rad
Sourced in United States, Germany
The CFX384 Touch instrument is a real-time PCR detection system designed for high-throughput gene expression analysis and target quantification. It features a 384-well format and multiple detection channels for simultaneous fluorescence monitoring during thermal cycling.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Primescript first strand cdna synthesis kit, by Takara Bio (2 mentions)
Sybr premix ex taq tli rnaseh plus, by Takara Bio (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Maxima sybr green rox qpcr master mix, by Thermo Fisher Scientific (2 mentions)
V1 sequencing chemistry, by Illumina (1 mentions)
Novaseq system, by Illumina (1 mentions)
D1000 screentape, by Agilent Technologies (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
Superscript 4 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Random primer, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
8 protocols using cfx384 touch instrument
1
Quantification of Cytokine Expression in Murine Tumors
2
Single-Cell RNA Sequencing of Nuclei
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After the determination of the nuclei suspension concentration, the suitable volume of nonsorted and sorted nuclei was calculated for a target cell recovery of 6000 and 3000 nuclei, respectively.
Samples were loaded on the 10 × Genomics single-cell-A chip (PN-no. 120236) and run on the Chromium System (Chromium Controller GCG-SR-1, 10 × Genomics).
After droplet generation and reverse transcription, the cDNA was recovered and amplified for 12 cycles. The samples where run on a High Sensitivity D1000 ScreenTape on the TapeStation (Agilent Technologies) and measured on the Qubit to determine the cDNA concentration.
The two sequencing libraries for FACS and notsorted nuclei were prepared using the Chromium Single Cell 3′ v2 Reagent kit (10 × Genomics, cat no. 120236/37/62) according to the manufacturer's protocol (CG00052 Single Cell 3′ Reagent Kit v2 User Guide). The adapter-ligated fragments were quantified by quantitative polymerase chain reaction using the Library quantification kit for Illumina (KAPA Biosystems) on a CFX384 Touch instrument (Bio-Rad Laboratories) before cluster generation and 26 + 8 + 0 + 91 cycles of sequencing of the two libraries in one S1 flowcell using the NovaSeq system and v1 sequencing chemistry (Illumina, Inc.).
Samples were loaded on the 10 × Genomics single-cell-A chip (PN-no. 120236) and run on the Chromium System (Chromium Controller GCG-SR-1, 10 × Genomics).
After droplet generation and reverse transcription, the cDNA was recovered and amplified for 12 cycles. The samples where run on a High Sensitivity D1000 ScreenTape on the TapeStation (Agilent Technologies) and measured on the Qubit to determine the cDNA concentration.
The two sequencing libraries for FACS and notsorted nuclei were prepared using the Chromium Single Cell 3′ v2 Reagent kit (10 × Genomics, cat no. 120236/37/62) according to the manufacturer's protocol (CG00052 Single Cell 3′ Reagent Kit v2 User Guide). The adapter-ligated fragments were quantified by quantitative polymerase chain reaction using the Library quantification kit for Illumina (KAPA Biosystems) on a CFX384 Touch instrument (Bio-Rad Laboratories) before cluster generation and 26 + 8 + 0 + 91 cycles of sequencing of the two libraries in one S1 flowcell using the NovaSeq system and v1 sequencing chemistry (Illumina, Inc.).
3
RNA Isolation, cDNA Synthesis, and qPCR Analysis
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RNA was isolated using the RNeasy Micro Kit (Qiagen, Venlo, the Netherlands). Complementary DNA was synthesized using SuperScript™ II Reverse Transcriptase (Thermo Fisher Scientific) with poly(T) primers (Sigma‐Aldrich). Quantitative PCR (qPCR) was performed using Sybr™ Select Master Mix (Thermo Fisher Scientific) in the CFX384 Touch instrument (Bio‐Rad, Veenendaal, the Netherlands) with GAPDH as endogenous reference gene. Table S1 shows sequences of gene specific primers. The comparative Ct method was used as described by Wong and Medrano.19
4
Quantitative PCR Analysis of Angiogenic Factors
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The total RNA extraction was conducted from cancer cells using an RNeasy Plus Mini Kit (Qiagen, Germantown, MD, USA), and reverse transcription was conducted using a PrimeScript First Strand cDNA Synthesis Kit (Takara, Mountain View, CA, USA). The qPCR analysis was performed using SYBR Premix Ex Taq (Tli RNase H Plus) (Takara) and a CFX384 Touch instrument (Bio-Rad, Hercules, CA, USA). The primers used here were as follows: Ang1, FW 5′-CATTCTTCGCTGCCATTCTG-3′ and Rev 5′-GCACATTGCCCATGTTGAATC-3′; Ang2, FW 5′-TTAGCACAAAGGATTCGGACAAT-3′ and Rev 5′-TTTTGTGGGTAGTACTGTCCATTCA-3′. The PCR conditions used were as follows: initial denaturation was performed at 95 °C for 1 min; 35 cycles of denaturation (95 °C for 30 s), annealing (60 °C for 30 s), and extension (72 °C for 1 min) were performed; and a final extension at 72 °C for 1 min was performed. The detailed information about the primers and thermal conditions is provided in Table 2 .
5
Adipose Tissue RNA Extraction and qRT-PCR Analysis
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RNA from 30mg of minced and frozen adipose tissue samples (Table 2C Supplementary Table S1
6
Yeast Total RNA Extraction and cDNA Synthesis
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Total RNA from yeast cells was extracted using the standard hot phenol/chloroform method. The total RNA was treated with TURBO DNase (Thermo Fisher Scientific) following the protocol from the manufacturer. 1 µg of the DNase‐treated RNA was converted to cDNA with Superscript IV Reverse Transcriptase Kit (Invitrogen, USA) using gene‐specific primers based on the manufacturer’s instructions. Diluted cDNA (1:20) was used in a PCR with the GoTaq qPCR Master Mix (Promega) and run on a CFX384 Touch Instrument (Bio‐Rad).
7
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. cDNA was obtained from 1 ug of total RNA, using reverse transcriptase and random primers (Invitrogen) according to the manufacturer’s instructions. cDNAs (1 mL for each sample) were amplified by PCR. Primer sequences are listed (Table S1 ). GAPDH was amplified as an internal control. Thermal cycling parameters were: 95°C for 2 min, 35 cycles of 95°C for 30 sec, 52–60°C (depending on the Tm of each individual set of primers) for 1 min and 72°C for 30 sec. QRT-PCR was performed using SYBR Green PCR Master Mix (Invitrogen) using a CFX384 Touch™ instrument (Bio-Rad, Hercules, CA, USA). The expression level for each gene was normalized to that of the GAPDH housekeeping gene in the same sample.
8
RNA Extraction and RT-qPCR for Primary Adipocytes
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For primary adipocytes, RNA was extracted with trizol and purified by using a column-based extraction kit (Zymo Research, Freiburg, Germany). From 50 mg of ground tissue, total RNA was extracted with 500 µl trizol and homogenized in the bullet blender for 5 min at 4°C followed by centrifugation for 10 min at 4°C and 12 000 g to separate the lipid layer. Phase separation was initiated by adding 100 µl chloroform. After 15-min centrifugation at 12 000 g and 4°C, RNA was precipitated from the upper clear phase with 250 µl of isopropanol. Lastly, RNA was purified with 500 µl of 75% ethanol, dried and solubilized in 30 µl DEPC water. Purified RNA was reversely transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Dreieich, Germany). Quantitative real-time PCR was performed using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific, Dreieich Germany) on a CFX384 Touch instrument (Bio-Rad, München, Germany). Primers were designed as intron spanning sequences to specifically amplify cDNA (Table S1).
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