Fig 1.
A comparison of small RNA library preparation workflows.
A) The traditional approach with unmodified adapters which results in tagged library and adapter dimer. B) The modified adapter approach which results in primarily tagged library.
Fig 2.
Ligation screen for modified adapters that suppress adapter dimer formation.
Example of modifications screened on the 5´adapter for ligation suppression against the Luo 3΄ Adapter with MP (n-1). Unmodified adapters were shown for comparison (U = unmodified). Adapter concentrations were 1 μM. Ligations performed with 10 U T4 RNA Ligase 1, 1 mM ATP, and 20% PEG, incubated for 2 hours at 37°C. Candidate modifications which reduce dimer formation are highlighted with blue box.
Fig 3.
Optimization of the 3´ adapter ligation step.
Synthetic Let-7d-5p (NNN) miRNA was ligated to the 3´ adapter using the same ligation conditions as the CleanTag library prep workflow step 1. A) Yield increase with addition of PEG 8000 using T4 RNA Ligase 2, truncated KQ and modified 3´ adapter (MP (n-1)). B) Specificity comparison between ligases used in 3´ ligation step: 1) T4 RNA Ligase 2, truncated; 2) T4 RNA Ligase 2, truncated KQ; 3) T4 RNA Ligase 1; 4) No Ligase. Both unmodified and modified (MP (n-1)) 3´ adapters were tested. Side products indicated with red arrows.
Fig 4.
Screen for the best combination of modified adapter pairs for suppression of adapter dimer.
Top combinations of modified adapters were tested in a full CleanTag library prep workflow from ligation to RT-PCR for dimer suppression. 0.7 ng Let-7d-3p (NNN) synthetic miRNA input. Samples run on a 4% agarose gel stained with ethidium bromide. Best combinations are shown in red boxes. U = unmodified.
Fig 5.
Investigation of modified adapter combinations at lower RNA inputs.
Brain total RNA at 1000 or 100 ng input was tested with candidate modified adapters in a full library preparation workflow. The modified 3´ adapter was MP (n-1) and the modified 5´ adapter was either 2´ OMe (n) or 2´ OMe (n-2). Agarose gel analysis of the product from 12 cycles of PCR. No adapter dilutions were made.
Fig 6.
Next-generation sequencing run comparing top modified adapter combinations.
Libraries prepared with unmodified or modified (MP(n-1)) 3´ adapter and unmodified or various modified 5´ adapter (1 = OMe(n), 2 = MP (n-1), or 3 = Ps(n)) using a pool of 963 synthetic miRNA (Miltenyi) following CleanTag library preparation protocol. Data analysis performed by TSRI. A) Agarose gel analysis of crude library PCR products. Sequencing Data: B) Average number of adapter dimer reads, C) Average number of filtered reads.
Fig 7.
Effect of adapter modifications on tagged library population.
Libraries were prepped with 1000 ng human brain total RNA and the CleanTag library prep protocol or recommended manufacturers conditions for Illumina and NEB kits. Data analysis performed by TSRI. A) Correlation plot of unmodified adapters and modified CleanTag adapters within the CleanTag library prep. Tagged miRNA are plotted after Log2 transformation. B) Venn diagram of CleanTag kit, Illumina TruSeq kit, and NEBNext kit depicting number of brain miRNA identified in all 3 replicates for each workflow.
Fig 8.
NGS data comparison between CleanTag and TruSeq Small RNA Library Preparation Kit.
Libraries prepared with TruSeq Small RNA Library Preparation Kit or CleanTag workflow with total human brain RNA input and gel purification. Samples sequenced on a HiSeq 2500 SR, 1x 100bp. Human total brain RNA at A) 100 ng, or B) 10 ng input. Data analysis performed using Geneious. Statistical analysis performed with GraphPad-One way ANOVA Turkeys multiple comparison test.
Fig 9.
Agarose gel analysis of PCR purified libraries with various total RNA inputs at 10ng.
Libraries prepared with CleanTag workflow. UHR is Universal Human Reference RNA.
Fig 10.
Comparison of crude and bead purified libraries using CleanTag or TruSeq small RNA library prep kit.
Bioanalyzer traces of libraries prepared using 1000 ng human total brain RNA input. Crude or AMPure XP purified PCR products with A) TruSeq small RNA library preparation kit, or B) CleanTag small RNA library preparation kit.
Fig 11.
Comparison of NGS data between gel purified and bead purified samples within a CleanTag workflow.
Libraries prepared with CleanTag small RNA library prep kit and human brain total RNA input. PCR samples were purified by gel extraction or 2-step AMPure XP bead-based protocol. Data analysis was performed with Geneious.
Fig 12.
Single cell quantities of small RNA can be tagged for next-generation sequencing.
Example (one of three replicates) bioanalyzer traces of crude PCR product libraries prepped using the CleanTag library prep workflow with human brain total RNA at A) 100 pg for 24 PCR cycles, or B) 10 pg inputs for 27 PCR cycles. C) Gel purified pool of ultra-low input (3 replicates of 100 pg and 3 replicates of 10 pg) samples.
Fig 13.
Next-generation sequencing data with single cell quantities of small RNA.
NGS data analysis of samples prepared with human brain total RNA inputs at 1000ng, 1ng, 100pg, and 10pg. 1000ng and 1ng samples were bead-purified and 100pg and 10pg samples were pooled and gel purified. Data analysis was performed using Galaxy. A) Raw read counts of total reads, filtered reads (after 3´adapter trimming and quality filtering), and adapter dimer reads. B) Normalized mapped read counts for small RNA types: miRNA, piRNA, other small RNA.
Fig 14.
Distribution of tagged small RNA in ultra low input libraries.
Small RNA libraries were prepared with modified adapters using various amounts of human brain total RNA input, sequenced on a HiSeq2500, and analyzed using BaseSpace sRNA App. The abundant categories and small RNA categories were further subcategorized.