Figure 1.
Library preparation using the SMART method.
A) The protocol used either poly A+ (0.025–0.5 µg) or total (0.05–1.0 µg) RNA. B) First-strand cDNA synthesis, together with template switching and continuous replication to the end of the oligonucleotide. C) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. D) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. E) Illumina sequencing library preparation.
Table 1.
List of modified oligonucleotides used for SMART, CapSMART and Non-CapSMART.
Figure 2.
Library preparation using the ligation method.
A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of RNA oligomers. A total of six tags (Table 3: TAG02, TAG04, TAG05, TAG06, TAG07, TAG12) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor, collection of biotinylated 5′ ends using beads, and sample pooling for multiplexing. H) Illumina sequencing library preparation.
Table 2.
List of modified oligonucleotides used for the ligation method.
Figure 3.
Library preparation using the CapSMART method.
A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides (Table 2: STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.
Figure 4.
Library preparation using the Non-CapSMART method.
A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Tobacco Acid Pyrophosphatase treatment to remove the 5′ cap structure, exposing a mono-phosphate group for subsequent ligation. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides (Table 2: STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.
Table 3.
List of modified oligonucleotides used for Illumina sequencing library preparation, and custom sequencing primers for the SMART and ligation methods.
Table 4.
Read numbers of sequences obtained from multiplexed samples using adult poly A+ RNA.
Figure 5.
Reproducibility of libraries generated by the SMART method using adult poly A+ RNA.
Plots showing correlation between sequence counts of six independent replicates (A: ID01 X 02; B: ID02 X 03; C: ID03 X 04; D: ID04 X 05; E: ID05 X 06; F: ID06 X 01). The results show a very high correlation (R = 0.99633–0.99996), indicating that the library preparation method is highly reproducible.
Figure 6.
Reproducibility of libraries generated by the ligation method using adult poly A+ RNA.
Plots showing correlation between sequence counts of six independent replicates (A: TG02 X 04; B: TG04 X 05; C: TG05 X 06; D: TG06 X 07; E: TG07 X 12; F: TG12 X 02). The results show a relatively low correlation (R = 0.09689–0.72684), indicating low reproducibility of the library preparation method.
Figure 7.
Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 7574000 and 7580000 on chromosome 2L.
Only the sequences mapped on the minus strand are depicted. Gene locations (Rapgap1 and CG13791) are depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.
Figure 8.
Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 4949000 and 4956000 on chromosome 2L.
Only the sequences mapped on the minus strand are depicted. Gene locations (Jon25Bi, Jon25Bii, Jon25Biii, and jet) are depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.
Figure 9.
Frequency distribution of mapped sequence reads on the Drosophila melanogaster genome (Release 5) between nucleotide positions 8041000 and 8044000 on chromosome 2L.
Only the sequences mapped on the minus strand are depicted. Gene location (RpL36A) is depicted at the bottom of the figure. Plots from three libraries using SMART and ligation methods {adult (TG02, TG04, ID01, and ID02) and embryo RNA} and from three libraries using CapSMART and Non-CapSMART methods (ID01, ID02, ID03, ID04, ID05, and ID06) are depicted in the figure.