However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can
manufacturer’s instructions and employed partial runs with a manifold to separate samples. To assemble our transcriptomes, we used GS De novo Assembler v2.3 (“Newbler”; 454 Life Sciences/Roche Branford, CT USA) set to default threshold options, and using the –vt option to remove adapters. ...
Both reactions were conducted in a realplex4 (Eppendorf, Germany) according to the manufacturer's instructions. Amplicon size was about 100 bp. All primers used for qPCR are listed in Table 2. We used negative controls without reverse transcriptase to check for remaining DNA contamination. To ...
The sizes of RNAs determined by northern blot analysis were sometimes larger than the sizes of the respective cDNAs determined by transcriptome sequencing (GmMuc4-like, GmP22), suggesting that in some cases we did not obtain full-length sequences, probably due to difficult assembling of the repet...
Considering that many gene-rich micro-chromosomes are still not fully assembled and that some chromosomes such as chr16 contain highly polymorphic regions [11], the number of annotated genes may increase further once all the micro-chromo- somes are fully assembled and polymorphic regions are ...
dsRNA synthesis. Double-stranded RNA (dsRNA) was synthesized from DNA templates amplified from cDNA of male and female adult worms, using the specific primer sequences indicated below, all of them contain- ing in addition a 17-nt T7 RNA Polymerase Promoter sequence at the 5′-end. For ...
Cells were harvested using trypsin (Hyclone), and total RNA was isolated using FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme), according to manufacturer’s instructions. RNA was converted to cDNA using oligo-dT primers (HiScript II Q RT SuperMix for qPCR, Vazyme). Quantitative PCR react...
The cDNAs for the second, third, and fourth cDNA libraries were synthesized using total RNAs derived from the aerial part of the chrysanthemum with a Mint-2 cDNA synthesis kit (evrogen; http://evrogen.com/) according to the manufacturer’s instructions. The first strand cDNAs were synthesized ...
Further, according to various embodiments described herein, processor-executable instructions for assembling and synthesizing nucleic acids. Thus, in some aspects the invention includes non-transitory computer-readable storage media encoded with instructions, executable by a processor, for generating assembled...
Vectors for use in eukaryotic host cells may require the addition of 3′poly(A) tail if the cDNA lacks poly(A). Additionally, the vector may contain promoters or enhancers that increase gene expression. Many promoters are known and used in the art. Most promoters are host specific and ...