MB49 or Biu-87 were cultured on dish or 2D laminin/collagen gels for 5 days. Cells were then harvested and total RNA was extracted using RNA Extraction Kit (Thermo Fisher, MA, USA) according to the manufacturer’s instructions. Reverse transcription of total RNA was performed using cDNA synthesis kits (Takara Bio, Tokyo, Japan) following the manufacturer’s instructions. PCR was performed with SYBR Green Supermixes (Biorad, MA, USA). Primer sequences were downloaded from https://pga.mgh.harvard.edu/primerbank/ .
Sybr green supermixes
Manufactured by Bio-Rad
Sourced in United States
SYBR Green Supermixes are pre-formulated reaction mixes designed for real-time quantitative PCR (qPCR) applications. The mixes contain SYBR Green I dye, necessary PCR components, and a thermostable DNA polymerase.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Iscript cdna synthesis kit, by Bio-Rad (4 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
7500 real time pcr machine, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Mircute plus mirna first strand cdna kit, by Tiangen Biotech (2 mentions)
Gdna eraser, by Takara Bio (2 mentions)
Rna extraction kit, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Takara Bio (1 mentions)
Cfx384 touch real time pcr detection system, by Bio-Rad (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Trizol, by Takara Bio (1 mentions)
M mlv reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Mirna specific primer, by RiboBio (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Chip assay kit, by Merck Group (1 mentions)
Ab2647, by Abcam (1 mentions)
Lightcycler system, by Roche (1 mentions)
14 protocols using sybr green supermixes
1
RNA Extraction and qPCR Analysis
2
Analyzing Cucurbitadienol Synthase Expression
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Total RNA was isolated from selected organs from I. amara and Ci. lanatus when they were just fruiting. qPCR assays were performed with SYBR Green supermixes (Bio-Rad, Hercules, CA) on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Two-step amplification conditions were used: 30 s at 95 ° C, 40 cycles of 10 s at 95 ° C and 30 s at 58 ° C. After qPCR, a melt curve was generated by heating the samples from 55 to 95 ° C with a 0.5 ° C elevated gradient. Primers targeting the transcript of I. amara and Ci. lanatus cucurbitadienol synthase and the housekeeping genes (actin) were designed (supplementary table 6 , Supplementary Material online). The method was used to calculate the relative fold change in gene expression. All qPCR experiments were performed with three technical and three biological replicates for I. amara and five biological replicates for Ci. lanatus, and the mean ± SE of biological replications was used for generating the bar chart.
3
Transcriptional Analysis of S. aureus Virulence Genes
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S. aureus was incubated with 1 µg/mL PaEVs for 6 h at 37°C under the static condition as described above. The bacterial cells were harvested at 6,000 × g for 10 min and immediately kept in -80°C until use. Total RNA from bacterial cells was extracted by TRIzol reagent (Ambion, Waltham, MA) according to the manufacturer's instruction. The contaminated genomic DNA was degraded with DNase I (Takara Bio, Shiga, Japan), and TRIzol extraction was performed again. cDNA was synthesized from 1 µg total RNA using random primer and M-MLV reverse transcriptase (Invitrogen, Waltham, MA). The expression of ldh2 and pflB gene in S. aureus was measured by RT-qPCR using SYBR Green Supermixes (Bio-Rad, Richmond, CA). The expression of the housekeeping ftsZ gene (encoding for FtsZ cell division protein) was used as a reference for normalization (Sihto et al., 2014 (link)). Primer sequences are shown in Table 1 . The following thermal protocol was used: 95 °C for 5 min, followed by 45 cycles of amplification (95 °C for 30 s, 58 °C for 30 s and 72 °C for 40 s), and 72 °C for 5 min.
4
Quantifying Gene Expression by RT-qPCR
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Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA), converted to cDNA by iScript cDNA synthesis kit (Bio-rad, Hercules, CA). Real-time PCR was performed by using SYBR Green Supermixes (Bio-rad) and a 7500 Real-time PCR machine (Applied Biosystems, Foster City, CA). The reaction conditions consisted of: stage 1, 95 °C for 10 min; stage 2, 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, which were concluded by the melting curve analysis process. Fold changes of gene expression were calculated using the 2−ΔΔCt method. The primer sequences are listed in Table 1 . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
5
RNA Extraction and RT-qPCR Analysis
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Total RNA was isolated by Trizol reagent (Invitrogen, USA) according to manufacturer's protocol. After quantification, and the reverse-transcription reactions were conducted with random primers and an M-MLV Reverse Transcriptase kit (Invitrogen, USA). RT-PCR was conducted using SYBR Green Supermixes (Cat. 1708882, Bio-Rad, USA). β-actin was used as internal control for normalization. Each sample was analyzed in triplicate. Relative expression of tested genes was normalized and analyzed by the 2−ΔΔCt method.
6
SALL4 and miRNA Expression Analysis
7
Quantifying Gene Expression Levels
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Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA), reverse transcribed into cDNA by using iScript cDNA synthesis kit (Bio-rad, Hercules, CA). Real-time PCR was performed by using SYBR Green Supermixes (Bio-rad) and a 7500 Real-time PCR machine (Applied Biosystems, Foster City, CA). Fold changes of gene expression were calculated using the 2-ΔΔCt method. The qRT-PCR primers used were as follows: GTPCH1 forward primer: 5’-TTGGAAAGGTCCATATCGGT-3′, reverse primer: 5’-ATTGTGCTCGTCACGGTTCT-3′; DHFR forward primer: 5’-AAGAACGGAAACCTGCCCTG-3′, reverse primer: 5’-GCCTCCCACTATCCAAACCA-3′; Nrf2 forward primer: 5’-AGC ACACCCAGTCAGAAACCAG-3′; reverse primer: 5’-TCTACAAACGGGAATGTCG-3′; β-actin forward primer: 5′- CGAGCATTCCCAAAGTTCTACAGTG-3′, reverse primer: 5′- CTACATACTTCCGAAAACCAGGGG-3′. β-actin was used as an internal control.
8
ChIP-seq for H3K27ac Enrichment
9
KLF15 Occupancy on LINC00689 Promoter
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CHIP Assay Kit (17-295, Millipore) was used to verify the interaction between KLF15 protein and LINC00689 promoter. Briefly, cells were cross-linked with 1% formaldehyde for 10 min, then lysed and sonicated to obtain chromatin fragments. The chromatins diluted by CHIP solution were immunoprecipitated with KLF15 (1:200, ab2647, Abcam, Cambridge, UK) or IgG (1:100, ab172730, Abcam) antibody at 4 °C overnight with rotation. After reversing the cross-links, the complexes were purified and analyzed by qPCR using SYBR Green Supermixes (Bio-Rad). The primers of LINC00689 promoter were described in Supplementary Table 3 .
10
miRNA and Gene Expression Analysis
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For first strand cDNA synthesis, 2 μg total RNA were extracted using PrimeScript™ RT reagent Kits accompanied with gDNA Eraser (TaKaRa, code no. RR047A) and miRNAs were extracted using miRcute Plus miRNA First-Strand cDNA Kits (TIANGEN, code no. KR211). miRNAs were quantified by stem-loop RT-PCR [32 (link)]. Gene expression was analyzed by qPCR using SYBR green supermixes from Bio-Rad and CFX96 real-time system. Each experiment was performed in three biological replicates. The expression of miRNAs and genes were calculated through the 2-ΔΔC (t) method [53 (link)] with internal reference genes TBP and U6, respectively. Primers are listed in Additional file 11 : Table S7. One-way ANOVA was used for statistical analyses in Microsoft Excel.
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