TITLE: Homemade Tn5 transposome assembly and activity evaluation
AUTHORS: Wei Xu

[DESCRIPTION]
Tn5 Transposome Assembly and Activity Assessment.

[BEFORE_START]
This protocol is basically for homemade Tn5 assembly and activity evaluation in ISSAAC-seq. Tn5 activity is determined by bulk ATAC-seq experiments, which mimics the in situ tagmentation condition in ISSAAC-seq experiments.

Unloaded Tn5 is available from vendors such as Diagenode (C01070010-10) or Lucigen (Cat. No. TNP92110). We obtain Tn5 from a local vendor, supplied at approximately 0.5 μg/μl.  Also, there are many standard protocols for Tn5 purification, and you can produce your own Tn5 protein if needed. Here are some references for Tn5 purification:
1) Generation and Purification of pTXB1.Tn5 (protocols.io)
2) Picelli, Simone, et al. "Tn5 transposase and tagmentation procedures for massively scaled sequencing projects." Genome research 24.12 (2014): 2033-2040.
3) Soroczynski,J., et al., OpenTn5 Project: Open-source resource for robust and scalable Tn5 transposase purification and characterization. Molecular Biology of the Cell, 2023. 34(2): p. 1026-1026.

[GUIDELINES]
This protocol is suitable for the assembly of purified Tn5 protein with a concentration between 0.5~1 μg/μl.

[STEPS]
SECTION: Reagent setup
38. Mix 25ul ME_A (100μM) with 25ul ME_Bottom (100μM) in a PCR tube  → 50ul Annealed ME_A (50μM) Mix 25ul ME_B (100μM) with 25ul ME_Bottom (100μM) in a PCR tube  → 50ul Annealed ME_B (50μM)
SECTION: Reagent setup
38. Annealing: by denaturing the oligos at 98oC first, and then slowly lowering the temperature from 98oC to 25oC, ME_A or ME_B will gradually anneal with ME_Bottom.  Put the oligo mixtures into a PCR thermocycler, run the following program: Temperature	Time	Number of cycles 98oC	5min	1 98oC	Decrease the temperature by 0.5oC at every 30 seconds 	50 73oC	Decrease the temperature by 0.5oC at every 30 seconds 	96 16oC	∞	1  (Alternatively, you can incubate the tubes in ~2 liters of boiling water, and let the ~98oC water slowly cool down to room temperature; or incubate them in a 98oC heating block and turn off the heating block to allow it to slowly cool down to room temperature.)
SECTION: Reagent setup
38. Mix 50 ul of 50 μM annealed ME_A and 50 ul of 50 μM annealed ME_B  → 100 ul of annealed ME_A + ME_B (50 μM total; 25 μM each)
SECTION: Reagent setup
38. 20 ul of annealed ME_A + ME_B (50 μM total; 25 μM each) + 30 ul H2O → 50 ul of annealed ME_A + ME_B (20 μM total; 10 μM each)
SECTION: Reagent setup
38. Assemble the Tn5 enzyme and annealed oligos:  Component	Amount Tn5 (L)	~48 ul Annealed ME_A + ME_B (20 μM total; 10 μM each) 	24 ul Coupling Buffer	88 ul Total	~160 ul  Component	Amount Tn5 (H)	~48 ul Annealed ME_A + ME_B (20 μM total; 10 μM each) 	24 ul Coupling Buffer	88 ul Total	~160 ul
SECTION: Reagent Setup
2. Reagent Setup
SECTION: Assemble Tn5
38. 
SECTION: Evaluate the activity of  Tn5
38. 
SECTION: Reagent Setup
2.2. Prepare Coupling Buffer:
     Component        Stock concentration        Concentration        For 1 ml          Tris-HCl, pH=7.5        1 M        50 mM        50 μl          NaCl        5 M        100 mM        20 μl          EDTA, pH=8.0        0.5 M        0.1 mM        0.2 μl          Triton X-100        10%        0.1%        10 μl          DTT        1 M        1 mM        1 μl          Glycerol        100%        50%        500 μl          H2O        -        -        412 μl     
 Store at -20 °C for up to 6 months.
SECTION: Tn5 transposome Assembly
3. Adapter annealing.
SECTION: Tn5 transposome Assembly
3.1. Mix 25 µL ME_S5 oligo (100 μM) with25 µL ME_Bottom oligo (100 μM) in a PCR tube, and label as "S5_adapter".
SECTION: Reagent Setup
2.3. Prepare oligonucleotides：
        #name  seq (5'-3')    ME_S5  TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    ME_S7  GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG    ME_bottom  Phos-C*T*G*T*C*T*C*T*T*A*T*A*C*A*C*A*T*C*/iInvdT/  
Dissolve the oligos in annealing buffer from above, to a final concentration of 100 μM. Store at -20 °C for up to 6 months.
SECTION: Tn5 transposome Assembly
3.5. Assemble the Tn5 enzyme and annealed oligos:
 
Incubate at room temperature for 60 min.  Store at -20 °C  for up to 1 year.

SECTION: Tn5 transposome Assembly
3.2. Mix 25 µL ME_S7 oligo (100 μM) with 25 µLME_Bottom oligo (100 μM) in a PCR tube, and label as  "S7_adapter".
SECTION: Tn5 transposome Assembly
3.4. Dilute the S5_adaptor (50 micromolar (µM)) and S7_adaptor(50 micromolar (µM)) with nuclease-free water to(20 micromolar (µM)). Continue to Step 8 or store at -20 °C  for up to 6 months.
SECTION: Tn5 transposome Assembly
3.3. Anneal adaptor oligo mixtures in a Thermocycler with the following reaction:
98 °C 3 min
slowly cooled to 16 °Cwith a temperature ramp of −0.1 °C/s.
16 °C hold.
SECTION: Tn5 protein concentration determination by Western blot.
1. The concentration and purity of in-house purified Tn5 protein could be further confirmed by Western blot before you start assembly. Tn5 protein is about 53 kDa, and its target band is between 55 kDa and 70 kDa markers on the gel. 
SECTION: Tn5 protein concentration determination by Western blot.
1.1. Prepare BSA protein concentration standards: dilute 3 µL of 2 mg/ml BSA standard with 21 µL of water to a final concentration of 0.25 mg/ml. 
SECTION: Tn5 protein concentration determination by Western blot.
1.2. Prepare BSA standards for WB：

 

Incubate at 95 °C for5 min, centrifuge briefly /. 

SECTION: Tn5 protein concentration determination by Western blot.
1.3. For each batch, mix 2 µL Tn5, 4 µL 5x loading buffer, and14 µL water. Incubate at 95 °C for5 min, centrifuge briefly /. 
SECTION: Tn5 protein concentration determination by Western blot.
1.4. Load the samples on a SDS-PAGE gel. After electrophoresis, the protein was transferred to NC membrane and stained with Ponceau S. Compare Tn5 with BSA standards to determine protein concentration. 
Expected result 1
 
Expected result 2
 

SECTION: Tn5 activity evaluation
4. Perform a fast ATAC-seq experiment to determine the activity of assembled Tn5.
SECTION: Reagent Setup
2.4. Prepare 4x THS TD Buffer:

 

SECTION: Reagent Setup
2.5. Prepare 0.1% (wt/vol) digitonin: Mix 0.5 µL 2% (wt/vol) digitonin stock with 9.5 µL nuclease-free water.
CRITICAL Make fresh. The Promega 2% (wt/vol) digitonin stock is dissolved in DMSO. Thaw at RT prior to use.
SECTION: Tn5 activity evaluation
4.1. Pellet 50,000 cells for each sample (#1 illumina Tn5，#2 Tn5_batch1, #3 Tn5_batch2…）at 1,000g, 4 °C for 3 min in a fixed-angle centrifuge.
SECTION: Tn5 activity evaluation
4.2. Aspirate and discard all supernatants, resuspend cell pellets in 1 mL ice-cold 1× DPBS–0.5% (wt/vol) BSA and centrifuge at 1,000g, 4 °C for 3 min.
SECTION: Tn5 activity evaluation
4.3. Aspirate all supernatants using a P1000 pipette, and briefly centrifuge again to collect leftover buffer from the bottom of the tube. Remove traces of the buffer using a P200 pipette with a 10 μL tip fitted on top of a 200 μL tip.
SECTION: Tn5 activity evaluation
4.4. Prepare 50 µL tagmentation mix for each sample by combining and mixing the reagents listed in the table below:

 

SECTION: Tn5 activity evaluation
4.5. Resuspend the cell pellet in 50 µL tagmentation mix by pipetting up and down 25 times.
SECTION: Tn5 activity evaluation
4.6. Put the 50 µL reaction on a thermomixer, and incubate at 37 °C, 800 rpm for 30 min.
SECTION: Tn5 activity evaluation
4.7. Cleanup reaction with a Zymo DNA Clean and Concentrator-5 kit, elute DNA in 21 μL of elution buffer.
SECTION: Tn5 activity evaluation
4.8. Set up the following PCR reaction:

 

SECTION: Tn5 activity evaluation
4.9. Take out 9 µL of each reaction, and mix with1 µL 10X SYBR Green and perform a qPCR analysis to decide the cycle number.
SECTION: Tn5 activity evaluation
4.10. Use the following cycling condition to perform a qPCR analysis, and monitor the amplification curve in linear scale.

 

 


 

SECTION: Reagent Setup
2.1. Prepare Annealing Buffer:
     Component      Stock concentration      Final concentration       For 1 ml        Tris-HCl, pH=8.0      1 M      10 mM      10 μl        NaCl      5 M      50 mM      10 μl        EDTA, pH=8.0      0.5 M      1 mM      2 μl        H2O      --      --      978 μl   

 Store at -20 °C for up to 6 months. 
SECTION: Tn5 activity evaluation
4.11. Validation of ATAC-seq library fragment length distribution.

Amplify the rest 11 μL PCR reaction for 12 cycles, purify the PCR product using 1.2x VAHTS DNA Clean Beads, elute in 20 μL water and check the size distribution using a bioanalyzer.