Figure 1.
Overview of RNA-seq library construction.
mRNA is purified from total RNA and fragmented to the desired size range. Next, the sheared RNA is reverse-transcribed to cDNA to form a DNA/RNA hybrid. The double-stranded cDNA is then synthesized, end-repaired and adenylated. Illumina adaptors are ligated to the processed double-stranded DNA and size selected. Finally, the size-selected ligated DNA products are amplified using primers to produce a sequence-ready library.
Figure 2.
Enforcing strand specificity using dUTP.
dTTP is substituted with dUTP during second strand cDNA synthesis. Y-shaped (partial-complementary) adapters are ligated and the dUTP-marked strand is digested with uracil-DNA gylcosylase (UDG). PCR amplification of this single strand confers strand specificity.
Figure 3.
Correlation of RPKM calculated from NSS and SS protocols and coverage statistics.
(a) Scatter plot of log10 correlation of SS- and NSS-derived RPKM values. SS-derived RPKM values are calculated from the sense strand only. r is the correlation coefficient. (b) Scatter plot of log10 correlation of SS- and NSS-derived RPKM values. Derived RPKM values are calculated from both strands of SS-data (c) Coverage plot along average gene body from 5′ to 3′ calculated from both NSS and SS methods. The percent GC content is also plotted.
Figure 4.
Survey of anti-sense alignments.
(a) An example of read alignment showing NSS- and SS-derived data for rice gene Os07g36090. The alignment is visualized using IGV (www.broadinstitute.org/igv/). Red and blue colors designate the directionality of reads. (b) Line plot showing percentage of anti-sense reads aligned to the average rice gene body from 5′ to 3′ end. (c) Line plot showing percent T along average rice gene body from 5′ to 3′ end.
Figure 5.
Average multiplex read distribution.
Bar plot of the read distribution among the eleven indices used for this study. Y axis represents the average percentage of indexed reads relative to the total number of reads from each lane. X axis shows the index sequences. Data is averaged from six lanes of data with standard error shown.
Figure 6.
Determination of the background cutoff for RNA-seq data.
Y axis shows the number of reads that map to non-coding regions relative to the total bp of non-coding regions (NCR). The x-axis displays the fraction of NCRs. As shown, 99% of the regions denoted as non-coding have fewer than 0.0015 reads/bp.
Figure 7.
Distribution of reads mapping to expressed and non-expressed genes.
(a) Distribution of sense alignments and (b) anti-sense alignments. The RPKM values were calculated from three replicates. The frequency shows the number of gene models per bin (vertical bars). A smoothed curve is plotted. Genes with average RPKM equal to zero are not shown in the histograms.
Figure 8.
Pink boxes highlight functions that are based on genome sequence and annotation and are not dependent on experimental data. Blue boxes highlight functions executed for each experimental run.