MiRNA cloning and high-throughput sequencing termed miR-Seq stands alone as a transcriptome-wide approach Lonaprisan to quantify miRNAs with single nucleotide resolution. ligation efficiencies of over 95% for both 3�� and 5�� ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from total RNA samples2. enzyme for efficient 5�� adenylation10. The phosphate can be added by pre-treatment of the oligonucleotide with a polynucleotide kinase or ordered with the modification directly. The randomized dinucleotide at the 5�� end is important for minimizing the sequence preference which RNA ligases exhibit for certain end-end joining reactions over others6. The 3�� dideoxycytosine places a blocking element at the 3�� end of the oligonucleotide to inhibit the formation of linker-linker concatamers during subsequent ligation steps and also serves to block the 3�� end of the miRNA-Linker molecule formed at the end of the 3�� ligation reaction11. The oligonucleotide linker sequence chosen for the unbiased miR-Seq method is as follows: RNA ligase nuclease-free H2O to a final volume of 40 ��l. Incubate reaction at 65 ��C for 1 hr. Terminate reaction by heating to 85 ��C for 5 min. Precipitate adenylated linker by the addition of 2.5 volumes of 100% ethanol and 1/3 volume of 10 M ammonium acetate to remove residual ATP and to prevent unanticipated ligations in future reactions. Briefly mix the alcohol salt and oligo solution to homogeneity spinning at full speed in a microcentrifuge (~15 0 x g) at 4 ��C for 20 min. Wash the pellet in 70% ethanol air dry and resuspend in the appropriate volume of nuclease-free H2O to yield 50-100 ��M of adenylated oligo. Confirm adenylation by running an 18% polyacrylamide gel. Prepare the gel Lonaprisan by mixing the following together: 6.3 g urea 6.75 ml of 40% acrylamide and 1.5 ml of 10x TBE. Add H2O to 15 ml. Mix until urea is completely dissolved and add 150 ��l 10% (w/v) ammonium persulfate (APS) and 15 ��l tetramethlyethylenediamine (TEMED). Once Lonaprisan gel has set pre-run for 30 min at 24 V/cm of gel width with 1x TBE running buffer at room temperature. Once 30 min is up flush wells with running buffer. Mix 5 pmol of adenylated oligonucleotides with and equal volume of 2x denaturing RNA loading buffer (95% (v/v) Formamide 0.02% (w/v) SDS 0.02% bromophenol blue (w/v) 0.01% (w/v) Xylene Cyanol 1 mM EDTA) heat briefly to 75 ��C and snap cooled on ice. Dispense entire sample into the well of the gel. Also include an identical amount of non-adenylated control and low molecular weight size standards. Run the gel Lonaprisan at 24 V/cm until the bromophenol blue is of the way to the bottom of the gel. Disassemble apparatus post-stain gel with 1x sybr-gold solution in 1x TBE and visualize on UV-transilluminator box (Figure 2). Figure 2 Adenylation of 3�� Linker with RNA-Ligase NOTE: Gel purify the adenylated oligo in a Rabbit Polyclonal to DOK4. manner similar to that described above if ��no input RNA�� controls are resulting in a high amount of PCR product as compared to samples with input RNA. 2 Linker Ligation to 3�� End of miRNA 3 Ligation Assemble the following components on ice in the order listed: 1.0 ��l 50% (w/v) PEG 8000 0.5 ��l 10x RNA ligase buffer (500 mM Tris- HCl pH 7.5 100 mM MgCl2 10 mM DTT) 1 ��l adenylated 3�� Linker (100 ��M) 0.5 ��l RNase inhibitor 0.5 Lonaprisan ��g total RNA 0.5 ��l T4 RNA ligase 2 nuclease-free H2O to a final volume of 5 ��l. Mix the reaction by pipetting as the solution is too viscous to vortex due to the high concentration of PEG 8000. Once thoroughly mixed place the reaction in a thermal cycler with a heated lid. Set the block to 16 ��C and incubate for 4 hr. NOTE: The reaction may be scaled up to a final volume of 20 ��l without any loss in ligation efficiency. React for 4 hr at ambient temperature or overnight at 4 ��C to yield essentially identical ligation efficiencies. Gel Purification Following 3�� ligation mix the reaction with an equal volume of 2x RNA loading buffer. Heat to 70 ��C for 5 min and snap cool on ice. Run the sample on a 15% polyacrylamide gel for PAGE purification. Prepare a 15% polyacrylamide gel containing 7 M urea in a fashion similar to that described.