Basal cell sorting was performed by disaggregating ∼200 Tg(ΔN-p63:Gal4, UAS:GFP) transgenic 2.5–3-dpf larvae into a single cell suspension as described previously (Bertrand et al., 2007 (link)). In brief, larvae were anesthetized using 0.2 mg/ml tricaine (Sigma-Aldrich) in E3, dissociated using Liberase TM (Roche; 13 U/ml, 15 min at 32°C.), and disrupted mechanically with a pestle. Cell suspensions were passaged through a 40-µm nylon mesh and washed twice in FACS buffer (centrifugation at 250 g for 5 min). Cell sorting of GFP-positive cells was performed on a FACS (Aria III; BD) using 488-nm excitation and 530/30-nm emission wavelengths. mRNA was extracted with oligo (dt)25 Dyna Beads (Invitrogen), followed by cDNA synthesis with RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific), and PCR Phire Hot Start II DNA polymerase (Thermo Fisher Scientific) using the following gene primers (PCR cycle numbers referred to in Fig. S4 b are indicated below):
Pcr phire hot start 2 dna polymerase
Manufactured by Thermo Fisher Scientific
Sourced in Lithuania
PCR Phire Hot Start II DNA polymerase is a thermostable DNA polymerase designed for high-fidelity PCR amplification. It features a hot-start mechanism that provides enhanced specificity and sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Aria 3, by BD (1 mentions)
Dynabeads oligo dt 25, by Thermo Fisher Scientific (1 mentions)
Liberase, by Roche (1 mentions)
Tricaine, by Merck Group (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Puretaq ready to go pcr bead, by GE Healthcare (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
2 protocols using pcr phire hot start 2 dna polymerase
1
Basal Cell Sorting and Transcriptome Analysis
2
Reverse Transcription PCR for Virus Sequencing
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For PCR analysis, cDNA was diluted 10-fold to minimize the concentration of possible compounds that may inhibit PCR reactions. PCR was carried out using puReTaq Ready-To-Go PCR Beads (GE Healthcare UK Limited), or Phusion DNA polymerase or PCR Phire Hot Start II DNA polymerase (Thermo Scientific, Vilnius, Lithuania), and virus-specific primers (S2 Table ). PCR products were purified using the E.Z.N.A. Gel Purification Kit (Omega BioTech Inc., Norcross, GA, USA) or QIAquick Gel Extraction Kit (Qiagen). They were sequenced by Macrogen (The Netherlands) or in the sequencing facility at Luke-Jokioinen. Most samples were sequenced directly without cloning, but a few PCR products were cloned into the vector pJET (Thermo Scientific) for sequencing using standard methods.
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