Terminator-treated samples were G/I-tailed by using 1,200 Units of yeast poly(A) polymerase (PAP) (Affymetrix), 0.5 mM GTP, 0.15 mM ITP, and incubated at 37°C for 90 minutes. Samples were diluted to 100 μL with water and G/I-tailed RNA was extracted with two sequential organic extractions (PCA followed by chloroform). The final aqueous layer was removed, and 10 μL of 3 M sodium acetate, 1 μL of GlycoBlue (Life Technologies), and 600 μL of 100% ethanol were added to the samples. Samples were incubated at −50°C for > 1 hour. Samples were pelleted by centrifugation for 30 minutes at 15,000 rpm at 4°C. Pellets were washed once in ~70% ethanol, and resuspended in 10 μL of water.
Yeast poly a polymerase pap
Manufactured by Thermo Fisher Scientific
Yeast poly(A) polymerase (PAP) is an enzyme that catalyzes the addition of a poly(A) tail to the 3' end of messenger RNA (mRNA) molecules. This process is essential for the stability and translation of mRNA in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Glycoblue, by Thermo Fisher Scientific (2 mentions)
Gotaq green master mix, by Promega (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Las4000, by GE Healthcare (1 mentions)
Gelred, by Biotium (1 mentions)
Amersham imager 680, by GE Healthcare (1 mentions)
Amersham imager, by Cytiva (1 mentions)
4 protocols using yeast poly a polymerase pap
1
RNA Extraction and G/I-Tailing Protocol
2
RNA Extraction and G/I-Tailing Protocol
3
Poly(A) Tail Length Assay
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The PAT assay was performed as described previously (Mishima and Tomari, 2016) (link). Briefly, 150 ng of total RNA was incubated with 75 U of yeast poly(A) polymerase (PAP) (Affymetrix) in the presence of GTP/ITP mix at 37°C for 60 min. cDNA was synthesized at 44°C for 15 min using the PrimeScript RT reagent kit with gDNA eraser (TaKaRa) and a y300 PAT universal C10 primer. PAT-PCR was performed using a 3´ UTR-specific forward primer and a y300 PAT universal C10 primer with GoTaq Green Master Mix (Promega).
The primer sequences are provided in Table S1. The PCR products were separated by 6% PAGE in 0.5×TBE. Gels were stained with GelRed (Biotium) and the signals were detected using LAS4000 (GE Healthcare) or Amersham Imager 680 (GE Healthcare). Signals were quantified using the "Plot profiles" function of the ImageJ software (http://imagej.nih.gov/ij/).
The primer sequences are provided in Table S1. The PCR products were separated by 6% PAGE in 0.5×TBE. Gels were stained with GelRed (Biotium) and the signals were detected using LAS4000 (GE Healthcare) or Amersham Imager 680 (GE Healthcare). Signals were quantified using the "Plot profiles" function of the ImageJ software (http://imagej.nih.gov/ij/).
4
Poly(G) Tailing-Based mRNA Profiling
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PAT assay was performed using a poly(G) tailing method (Kusov et al., 2001) with some modifications. Briefly, 150 ng of total RNA were incubated with 75 U of yeast poly(A) polymerase (PAP) (Affymetrix) in the presence of 0.375 mM GTP and 0.125 mM ITP at 37 C for 60 min. cDNA was synthesized using the PrimeScript RT reagent kit with gDNA eraser (TAKARA) and a customized PAT primer (y300 PAT universal C10: 5 0 -GGTAATACGACTCAC TATAGCGAGACCCCCCCCCCTT-3 0 ). cDNA synthesis was performed at 44 C for 15 min. PAT-PCR was performed using a gene-specific forward primer and y300 PAT universal C10 primes with GoTaq Green Master Mix (Promega). The control PCR was performed using a gene-specific forward primer and a reverse primer complementary to the annotated 3 0 end, followed by the sequence of the y300 PAT primer. Experiments were repeated multiple times to confirm reproducibility.
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