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The best hits were used to decide on sequence direction of transcripts.
If results of different databases conflict with each other, a priority order of nr, SwissProt and KEGG were followed to decide the direction of transcripts.
Since our transcriptomes are reconstructed from a non strand-specific RNA-seq dataset, we determined the direction of transcripts based on the splice junction sequences.
If hits of different databases conflicted with each other, a priority order of NR, SwissProt, KEGG and COG was followed for deciding the sequence direction of transcripts.
Next, Blastx search (E-value < 1E−5) against the NCBI nr, Swiss-Prot and KEGG databases was performed and the best aligning results were used to decide the direction of transcripts.
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We sampled 16 representative examples of these RNAs by quantitative RT-PCR to independently confirm the downregulation seen on the array; the direction of transcript level change (i.e. down) was confirmed in every case (Figure 2).
Interestingly, these tissues differ in the major direction of transcript level changes with age.
The transcription factor activity prediction tool in the Ingenuity Systems® software uses an algorithm that weighs the direction of transcript level changes in the data set of interest and an empirical database to elucidate the genes that are likely regulated by a given transcription factor.
We then determined the direction of transcription of these transcripts.
To confirm the direction of transcription of the putative antisense transcripts, we employed strand-specific primers in reverse transcription (RT) reactions to specifically target either sense or antisense transcripts.
One of the advantages of full-length cDNAs is that we can determine the splicing pattern and reading direction of the transcripts in the genome.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com