High quality total RNA was extracted and purified as previously described (Kenny et al. 2016 (link)). cDNA was generated by using SMARTer polymerase chain reaction (PCR) cDNA Synthesis kit (Clontech). Gene-specific primers were designed for each gene of interest (supplementary S5, Supplementary Material online). PCR products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA). Linearized template DNA (amplified from plasmid with T7/SP6 primers) was used to synthesize RNA probes with either T7 or SP6 RNA polymerase (Life Technologies), and DIG-labeling mix (Roche, Indianapolis, IN, USA). WISH protocol was performed, as previously described (Perry et al. 2015 (link)).
Smarter polymerase chain reaction
Manufactured by Takara Bio
The SMARTer polymerase chain reaction is a laboratory equipment used for DNA amplification. It functions by facilitating the exponential replication of specific DNA sequences through thermal cycling and enzymatic reactions.
Automatically generated - may contain errors
2 protocols using smarter polymerase chain reaction
1
Whole-mount in situ hybridization protocol
2
PacBio Full-length cDNA Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Equal quantities of RNA were extracted from five tissue samples, including root, trophophyll-S1, sporophyll-S1, trophophyll-S2, and sporophyll-S2, for the construction of a PacBio cDNA library. Briefly, 1 μg of RNA from the pooled RNA sample was reverse transcribed using the Clontech SMARTer polymerase chain reaction (PCR) cDNA Synthesis Kit and oligo (dT) to generate the first-strand cDNA. The second strand was then synthesized and amplified with the 5′ PCR primer to obtain full-length cDNA with barcodes. Size selection was carried out using the BluePippin Size Selection System protocol, with two size bins for the mixed sample: 1–4 kb and > 4 kb. Finally, the cDNA library was sequenced on the PacBio Sequal platform using a SMRT cell.
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