The, non-stranded, Illumina TruSeq kit can be adapted to generate strand specific libraries and can be used to access detailed information on the transcriptome.
However, consistent with the findings of Haddad et al. [ 70] our extensive attempts to generate strand specific amplicons were unsuccessful (data not shown), therefore a microarray approach was employed.
To generate strand-specific standard curves for ssqPCR, and strand RNA was transcribed in vitro from a plasmid containing a portion of the nsP1 gene from either ONNV or CHIKV.
Fragmented RNA was used to generate strand-specific RNA-Seq libraries according to the Directional mRNA-Seq Library Preparation Protocol (Illumina).
To generate Strand-seq data, cells are cultured with the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) for one round of DNA replication.
Therefore, we adopted this method for use in Plasmodium although alternative strategies to generate strand-specific libraries may also be well suited [ 20].
Briefly, 4 μg of DNase-treated total RNA from androgen-treated or control LNCaP cells were used to generate strand-specific cDNA.
ATP-hydrolysis could drive the h2i/ps1β inward to generate strand separation that is captured by the MSSB as shown in Figure 7 of (Froelich et al., 2014 ).
In order to generate strand-specific array probes, two separate PCR reactions were performed for each clone, in one case using a 5'-aminolinked primer for the forward strand, and in the other case, for the reverse strand.
To generate strand-specific RNA-seq libraries, the second strand of the gel-purified adapter-ligated cDNA containing uracil was digested enzymatically in 30 μl reaction volumes containing 1× Uracil-DNA Glycosylase buffer and 1 U Uracil-DNA Glycosylase (Bioline).
RNA-Seq was performed using the protocol developed in (Nagalakshmi et al. 2008), further described in (Nagalakshmi et al. 2010) and (Waern et al. 2011), and using the modifications developed by (Parkhomchuk et al. 2009) to generate strand-specific reads.
