TITLE: dU-Tn5 stranded RNA-seq experiment
AUTHORS: Xiaoyuan Tao

[DESCRIPTION]
In this protocol, we describe a novel Tn5-based stranded RNA-seq procedure, in which deoxy-UTP-labeled Tn5 (dU-Tn5) is applied in library construction to preserve the strand-specificity of transcripts.  The stranded library preparation section only contains 8 steps, which is straightforward and easy-to-do for library preparation. 




[STEPS]
SECTION: mRNA purification
4. mRNA was purified from 1 μg of total RNA using oligo(dT)-attached mRNA capture magnetic beads (Vazyme, cat#401) following the user manual. The final purified mRNA was dissolved in 10.5 μl ddH2O.
6. Second-strand cDNA was synthesized by adding 10 μl 5 x second strand buffer, 1 μl dUTP-containing dNTP mix (20 mM dUTP, 10 mM each dATP, dGTP, and dCTP), 1 μl E. coli DNA ligase, 2 μl DNA polymerase I, 0.06 μl RNase H, and 15.94 μl ddH2O to adjust the reaction volume to 50 μl. The solution was incubated at 16 °C for 1 h.
7. The resulting double-stranded cDNA from previous step was tagmented by adding 20 μl 5x Tn5 Tagmentation Buffer,16 μl 50% PEG 8000, 2 pmol(0.5 μl) Tn5 transposase, and 14 μl ddH2O to adjust the reaction volume to 100 μl. The tagmentation reaction was performed at 55°C for 10 min, after which 10 μl 0.2% SDS was added and the enzyme deactivated by heating to 85°C for 5 min. 
SECTION: Stranded library preparation
5. For first-strand cDNA synthesis, mRNA was reverse transcribed in a PCR tube with the following setup:10 μl purified mRNA, 1 μl 50 μM Oligo (dT)23VN, 1 μl 50 μM random primers, 4 μl 5 × HiScript III Buffer, 0.5 μl dNTP Mix (10 mM each dATP, dTTP, dGTP, and dCTP), 1 μl 40 U/μl Recombinant RNasin® Ribonuclease Inhibitor, 1 μl 200 U/μl Hiscript III Reverse Transcriptase, 1 μl 120 ng/μl actinomycin D, and ddH2O as needed to adjust the reaction volume to 20 μl. The reverse transcription was performed under the following program: using a heated lid, 25°C for 5 min, 55°C for 45 min, and finally 85°C for 5 min for deactivation. 

SECTION: dU-Tn5 assembly
3. Set up the following reaction in a 1.5 mL centrifuge tube: 5 μL of Tn5 transposase (10 pmol/μL), 1.2 μL of adaptor mix (50 pmol/μL), 6.3 μL of coupling buffer (included in the commerical Tn5 products). Pipette 20 times gently to mix well and incubate at 30°C in a water bath for 1 h. The final concentration of transposase = 4 pmol/ μL. Store at -20°C until use.
 


SECTION: dU-Tn5 assembly
1. Dilute oligos (Primer A, Primer B, and dU-Primer C, refer to Table S1) to 100 μM concentration using the annealing buffer.
SECTION: dU-Tn5 assembly
2. Set up the following two reactions in PCR tubes: Reaction 1 (adaptor AB), 10 μL of 100 μM primer A, 10 μL of 100 μM primer B; Reaction 2 (adaptor AC), 10 μL of 100 μM primer A, 10 of μL 100 μM primer C.Anneal the adapters in the PCR machine using the following program: heat lid (102°C), 75°C for 15 min, 60°C for 10 min, 50°C for 10 min, 40°C for 10 min, 25°C for 30 min.Combine adaptor AB and adaptor AC at 1:1 ratio, designated as“adaptor mix”. 
 


8. Add 100 μHieff NGS® Smarter DNA Clean Beadsto the tagmentation products, purified the DNA following the user guide.Dissolvethe resulting DNA in 20 μl ddH2O. 
9. Preparation for PCR amplification was then carried out by mixing the 20 μl of eluted DNA with 25 μl 2x Phanta® Max buffer, 2 μl dNTP Mix (10 mM each dATP, dTTP, dGTP, and dCTP), and 1 μl Bst 2.0, and extension was performed at 72°C for 20 min followed by deactivation at 85°C for 20 min. 
10. Add 1.5 μl Heat-labile UDG (1 U/μl), 1 μl Phanta® Max Super-Fidelity DNA Polymerase, 1 μl primer N50X (20 μM), and 1 μl primer N70X (20 μM)to the PCR tube, and PCR was performed according to the following program: 25 °C for 20 min; 95 °C for 3 min; 14 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; 72 °C for 5 min; 4 °C for 1 min. 
11. Take3 μl of the PCR productfor agarose gel electrophoresis to determine the concentration and size distribution of bulk DNA products.
 
  

12. The PCR products were purified using 60 μl (1.2 volume) Hieff NGS® Smarter DNA Clean Beads (Yeasen) following the user guide, and the resulting library was dissolved in 30 μl ddH2O for further quality control (QC) and NGS sequencing.