The DNAs for selection (Supplementary Table S1) and sensing were purchased from Integrated DNA Technologies (Coralville, IA, USA). The other DNAs were from Eurofins (Huntsville, AL, USA; Supplementary Table S2). The metal salts were from Sigma-Aldrich at the highest available purity. Tris(hydroxymethyl)aminomethane (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), ethylenediaminetetraacetic acid (EDTA), NaCl and ammonium acetate were from Mandel Scientific (Guelph, Ontario, Canada). SsoFast EvaGreen supermix was from Bio-Rad. T4-DNA ligase, dNTP mix, Taq DNA polymerase and DNA ladder were from New England Biolabs.
Dntp mix
Manufactured by New England Biolabs
Sourced in United States
The DNTP mix is a solution containing the four deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dTTP) necessary for DNA synthesis. This product is designed for use in various molecular biology applications requiring the incorporation of DNA nucleotides.
Automatically generated - may contain errors
Lab products found in correlation
Taq dna polymerase, by New England Biolabs (6 mentions)
Rox reference dye, by Thermo Fisher Scientific (6 mentions)
Rnase inhibitor, by New England Biolabs (5 mentions)
M mulv reverse transcriptase, by New England Biolabs (5 mentions)
Taq buffer, by New England Biolabs (4 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Qiaquick gel extraction kit, by Qiagen (4 mentions)
M mulv reverse transcriptase buffer, by New England Biolabs (3 mentions)
Bst 2.0 warmstart dna polymerase, by New England Biolabs (3 mentions)
Glycogen, by Thermo Fisher Scientific (3 mentions)
Sybr green, by Thermo Fisher Scientific (3 mentions)
Superscript 4 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
7 deaza dgtp, by New England Biolabs (2 mentions)
Random hexamer, by New England Biolabs (2 mentions)
Betaine, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Takara Bio (2 mentions)
Rnasin plus, by Promega (2 mentions)
Smartscribe, by Takara Bio (2 mentions)
Smart seq2 lysis buffer, by New England Biolabs (2 mentions)
Oligo dt30vn primer, by New England Biolabs (2 mentions)
47 protocols using dntp mix
1
Metal-Sensing DNA Sequence Protocol
2
Reverse Transcription and Real-Time PCR Protocol
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Total RNA was primed with 1 μl oligo dT (12–18) primer (50 μM) in a 6‐μl reaction system, and then incubated at 70°C for 10 min and rapidly cooled on ice prior to reverse transcription reaction. Reverse transcription was carried out at 42°C for 1 h in a final volume of 10 μl containing 2 μl of 5× M‐MLV buffer (TAKARA BIO), 0.5 μl of 10 mM dNTP mix (NEW ENGLAND Biolabs), 0.25 μl of RNase Inhibitor (TAKARA BIO), 1 μl of RTase M‐MLV (RNase H‐) (TAKARA BIO) followed by incubation at 70°C for 15 min to terminate the reaction and stored at ‐20°C until use. For real‐time PCR, the cDNA samples were diluted tenfold.
RT‐qPCR was conducted on LightCycler 96 (Roche). Each reaction mixture consisted of 2 μl of cDNA, 0.5 μl each of forward (10 μM) and reverse primers (10 μM), 12.5 μl 2× SYBR Premix Ex TaqTM II (Tli RNaseH Plus) (TAKARA BIO), and 9.5 μl of nuclease‐free water (Millipore) in a total reaction volume of 25 μl. The thermal cycler program consisted of 40 cycles of 95°C for 15 s and 60°C for 1 min. Primers were designed using Primer Premier 5.0 (PRIMIER Biosoft). The primer sequences are listed in TableS1 . Data were obtained from three replicate assays for each sample with normalization to ActB, and relative gene abundance was calculated using the method of 2−ΔΔCt as previously described.10
RT‐qPCR was conducted on LightCycler 96 (Roche). Each reaction mixture consisted of 2 μl of cDNA, 0.5 μl each of forward (10 μM) and reverse primers (10 μM), 12.5 μl 2× SYBR Premix Ex TaqTM II (Tli RNaseH Plus) (TAKARA BIO), and 9.5 μl of nuclease‐free water (Millipore) in a total reaction volume of 25 μl. The thermal cycler program consisted of 40 cycles of 95°C for 15 s and 60°C for 1 min. Primers were designed using Primer Premier 5.0 (PRIMIER Biosoft). The primer sequences are listed in Table
3
Reverse Transcription Protocol for cDNA Synthesis
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A total of 0.7 μl RT Primer (from a 100 μM stock) was added to the purified sample and incubated at 75°C for 3 min, 37°C for 10 min and 25°C for 10 min. A total of 4 μl of ProtoScript II Buffer, 1 μl dNTP mix (NEB), 0.2 μl 1 M MgCl2, 2 μl DTT (NEB) and 2 μl of ProtoScript II were added and the mixture incubated at 42°C for 12 h with a 105°C heated lid. (Alternatively, for standard conditions [see section on 2′-5′ linkages], no MgCl2 was added and the incubation was at 50°C for 1 h.) Ten microliter of water was added and the mixture was run through an Oligo Clean & Concentrator spin column, including the RNA degradation step as per the manufacturer's instructions. The cDNA was eluted in 30 μl of TE, pH 7 and stored at 4°C. The cDNA stock concentration was measured with a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific), and typically found to be in the fraction of a μg/μl range.
4
Termitomyces RNA Extraction and cDNA Synthesis
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We reanalyzed the relative expression levels of TC-related gene sequences in RNAseq data obtained from axenic Termitomyces sp. 153 and J132, as well as fresh and old fungus comb and nodules on which young workers feed21 (link). The transcript sequence of DS2 was obtained by PCR from a cDNA template. RNA of Termitomyces sp. T153 was extracted from frozen mycelium of a culture grown on PDA plate cultures for ~2 weeks using the “Isolate II RNA plant” Kit (Bioline). cDNA was obtained using the following protocol: 2 µg RNA, 2 µL Oligo d(T)23VN primer (NEB), 1 µL dNTP mix (25 mM each, biotech rabbit) and adjustment with water to a volume of 18 µL; incubation for 5 min at 65 °C, followed by addition of 6 µL RT buffer, 1 µL RT enzyme (Thermo, Maxima H Minus), 1 µL RNase-Out (Invitrogen) and 4 µL water; cDNA synthesis was performed for 2–3 h at 46 °C finalized by an inactivation step at 85 °C for 5 min.
5
Optimized mRNA and EV RNA Reverse Transcription
6
cDNA Synthesis and Second Strand Preparation
7
Transcriptional Profiling of C. difficile 630
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Total RNA was isolated from C. difficile 630 grown in BHIS at early exponential phase (t = 3 hours, OD600 = 0.4-0.5), late exponential phase (t = 5 hours, OD600 = 0.8-0.9), and stationary phase (t = 24 hours, OD600 = 0.6-0.8) using RNAprotect Bacteria Reagent (Qiagen) and the FastRNA Pro Blue Kit (MP Biomedicals LLC., Illkirch, France) in accordance with the manufacturer’s instructions. Genomic DNA was removed from total RNA samples using TURBO DNase (Life Technologies). Equal amounts of RNA were reverse transcribed into complementary DNA (cDNA) for expression analysis. Briefly, one μg random primers (Invitrogen) and 40 units RNasin Plus RNase inhibitor (Promega) was added to one μg RNA in a final volume of 10 μl, and incubated at 65°C for 10 min followed by room temperature for 30 min. The following first-strand mixture was added for cDNA synthesis: four μl of 5x first-strand buffer (Invitrogen), two μl 0.1 M DTT (Invitrogen), two μl 10 mM dNTP mix (New England BioLabs), and 1.5 μl Superscript II (Invitrogen). The reaction mixture was incubated at 25°C for 10 minutes, 42°C for 1 h, and finally 70°C for 15 minutes. RT-PCRs were performed with gene specific primers (Additional file 2 : Table S1) using cDNA as a template.
8
Single-cell transcriptomics of bone marrow
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Lineage-depleted bone marrow cells were obtained by crushing and stained with the following antibodies on ice for 30 min: CD41, CD45, CD51, CD61, CD71, CD200, Ter119 and VCAM1 (Table S4 ). Indexed single-cells were sorted into 5μl of Smart-Seq2 lysis buffer (2μM oligo-dT30VN primer, 2mM dNTP mix (10mM each, NEB), 1:50 RNAse inhibitor (promega) and 1:125 Triton X-100 10% (Sigma-Aldrich)) and immediately snap frozen in an ethanol and dry ice bath. Plates were kept at -80 °C until processing. cDNA amplification was performed using a modified Smart-Seq2 protocol by adding, after 3 min at 72 °C, 5μl of RT mix containing 1× SMART First Strand Buffer (Clontech), 2 mM dithiothreitol (Clontech), 2 μM template switching oligo (Exiqon), 10 U μl-1 SMARTScribe (Clontech) and 10 U μl-1 RNASin plus (Promega). Transcriptome amplification was performed using 1X KAPA HiFi HS MM and 0.1μM ISPCR primer, with 21 PCR enrichment cycles. Libraries were constructed using in house produced Tn554 (link) at 1:100 dilution and sequenced on an Illumina Next-Seq 500 sequencer, with 75 cycles single end sequencing.
9
Quantifying mRNA Levels in Myeloid Cells
10
Trichoplax Calcium Channel Genes
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The Trichoplax genome encodes single gene homologues for each of the three metazoan Cav channel types (Cav1, Cav2, and Cav3; NCBI accession nos. XM_002108894 .1, XM_002109739 .1, and KJ466205 , respectively), as well as a single Cavβ accessory subunit gene (XM_002110305 .1) and three Cavα2δ Cav1/Cav2 accessory subunit genes (Cavα2δa, Cavα2δb, and Cavα2δc; NCBI accession nos. XM_002112625 .1, XM_002112621 .1, and XM_002111347 .1). Primers were designed to amplify ∼500-bp cDNA fragments of each of these genes by RT–PCR (Table 1 ), using a cDNA library prepared by RT (SuperScript III Reverse Transcription; Thermo Fisher Scientific) with an anchored oligo-dT18 primer (Table 1 ) and whole-animal total RNA. PCR amplification was achieved in 25-µl reactions each containing 1.25 µl of 10 µM forward and reverse primers (Table 1 ); 0.125 µl of Taq DNA polymerase and 2.5 µl of corresponding 10× buffer (New England Biolabs, Inc.); 1 µl of 25 mM MgCl2; 0.5 µl of 10 mM dNTP mix (New England Biolabs, Inc.); and 0.5 µl of cDNA template. Thermocycling conditions were 95°C for 2 min, 30 cycles of 94°C for 1 min, 59°C for 45 s, and 72°C for 1 min, and a final 10-min extension at 72°C.
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