================= ATAC-see ================= .. warning:: ATAC-see is a very inefficient procedure, and gives effectively useless results compared to background, even in flow cytometry. Transposase buffer preparation --------------------------------- .. time:: 1.5 hours. 1. Order the following primers: ============ ====== ============= ================ ==================================== Primer name Scale Purification 5' modification Sequence ============ ====== ============= ================ ==================================== Tn5ME_rev 25nmol DSL Phosphate `CTGTCTCTTATACACATCT` Tn5ME_A 50nmol HPLC Cy5 `TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG` Tn5ME_B 50nmol HPLC Cy5 `GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG` ============ ====== ============= ================ ==================================== 2. Resuspend each primer separately to 100 uM (the standard stock dilution, nmol * 10 uL). 3. Prepare 1:1 solutions of Tn5ME_rev / Tn5ME_A (A+rev) and Tn5ME_rev / Tn5ME_B (B+rev). Each primer is now at 50 uM. 4. Assemble these double stranded oligos by heating to 95C for 5 minutes, followed by slow cooling to room temperature. To do this on a thermocycler, use a program that does ends after the denaturation step, instead of ending in a hold step. 5. Prepare 5 uM stock solution of Tn5/Cy5. Prepare this either from freshly purified Tn5 in dialysis buffer: ================= =============== Component Volume fraction ================= =============== A+rev primers 0.125 B+rev primers 0.125 Glycerol 0.4 Dialysis buffer 0.12 50 uM Tn5 0.1 DI H2O 0.13 ================= =============== If Tn5 is already stored in a 50% glycerol/50% 2x dialysis stock solution at 20 uM, use the following recipe: ================== =============== Component Volume fraction ================== =============== A+rev primers 0.125 B+rev primers 0.125 Glycerol 0.25 20 uM Tn5+Glycerol 0.25 DI H2O 0.25 ================== =============== ATAC-see -------- We need the following prepared buffers: * **Nuclear permeabilization buffer**: ================== ============== ==================== Component Concentration Amount/100 mL final ================== ============== ==================== Tris-Cl 10 mM 0.1576 g NaCl 10 mM 0.058 g MgCl2(anhydrous) 3 mM 0.0287 g Igepal CA-630 0.01% 10 uL HCl to pH 7.4 ================== ============== ==================== * **Wash buffer**: ================== ============== ==================== Component Concentration Amount/100 mL final ================== ============== ==================== PBS Base SDS 0.01% .01 g EDTA 50 mM 1.461 g ================== ============== ==================== * **2x TD buffer** (from `this nature paper `_): ============================== =============================== ============== Component Concentration Amount/L final ============================== =============================== ============== Tris 20 mM 3.264 g MgCl2 10 m 0.95 g DI H2O main solvent Acetic acid to pH 7.6, before DMF addition Dimethylformamide(DMF) 20% (v/v) ============================== =============================== ============== 1. After cell fixation, permeabilize cells with lysis buffer for 10 minutes at room temperature. 2. Wash with PBS twice. 3. Prepare transpose mixture: ================== =============== Component Volume fraction ================== =============== 2x TD buffer 0.5 5 mM assembled Tn5 0.02 DI H2O 0.48 ================== =============== 4. Add 50 uL of transpose mixture to cells to be transposed. Place the cells in a humid 37C box for 30 minutes. 5. For plated cells, wash with wash buffer three times, for 15 minutes each at 55C. For suspended cells, wash twice. 6. Add PBS media to cells and image/flow the resulting cells.