Journal: Scientific Reports
Article Title: Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer formation and bias correction
Figure Lengend Snippet: TGIRT-seq workflow and design of an improved R2R adapter that decreases adapter-dimer formation. ( A ) TGIRT-seq workflow. In the first step, TGIRT enzyme binds to an artificial template-primer substrate comprised of an RNA oligonucleotide containing an Illumina R2 sequence with a 3′-end blocking group (3SpC3) annealed to a complementary DNA oligonucleotide (R2R) that leaves a single nucleotide 3′ overhang, which can direct template-switching by base pairing to the 3′ end of an RNA template. For the preparation of TGIRT-seq libraries from pools of RNAs, the DNA primer consists of a mixture of DNA oligonucleotides that leave A, C, G, and T 3′ overhangs (denoted N). After pre-incubation of the TGIRT enzyme with the target RNAs and template-primer (see Methods), template-switching and reverse transcription of an RNA template are initiated by adding dNTPs. The resulting cDNA with an R2R adapter attached to its 5′ end is incubated with NaOH to degrade the RNA template and neutralized with HCl, followed by two rounds of MinElute clean-up using the same MinElute column (Qiagen). A pre-adenylated oligonucleotide containing the reverse complement of an Illumina R1 sequence (R1R) is then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA ligase (New England Biolabs), followed by MinElute clean-up and 12 cycles of PCR amplification with primers that add indices and capture sites for Illumina sequencing. Unused R2R adapters that are carried over from previous steps are also ligated to the R1R adapter by the 5′ App DNA/RNA ligase (New England Biolabs), resulting in the formation of adapter dimers (pathway at right), which are removed by AMPure beads clean-up prior to sequencing. ( B ) Taking into account known biases of the 5′ App DNA/RNA ligase 7 , 28 , 29 , the R2R adapter used previously in TGIRT-seq (denoted NTC) was modified by inserting a single T-residue at position −3, creating a modified R2R adapter (denoted NTT), which decreases adapter-dimer formation. ( C ) Bioanalyzer traces comparing adapter-dimer formation using the previous NTC and improved NTT R2R adapters. 2 pmole of the NTC or NTC R2R adapter was ligated to 40 pmole of adenylated R1R adapter followed by 12 cycles of PCR according to the TGIRT-seq protocol and 1 round of clean-up with 1.4X AMPure beads to remove salt, PCR primers, and adapter dimers. The products were analyzed by using a 2100 Bioanalyzer (Agilent) with a high sensitivity DNA chip. M: internal markers in the NTC (red) or NTT (blue) traces.
Article Snippet: The R1R DNA adapter was pre-adenylated by using an adenylation kit (New England Biolabs) and then ligated to the 3′ end of the cDNA by using thermostable 5′ App DNA/RNA Ligase (New England Biolabs) for 2 h at 65 °C.
Techniques: Sequencing, Blocking Assay, Incubation, Polymerase Chain Reaction, Amplification, Modification, Chromatin Immunoprecipitation