Preparation

Materials and Reagents

Equipment

Equipment

• Chilled Hammer
• DNA/RNA LoBind tubes (Eppendorf, cat. no. 022431021)
• Refrigerated centrifuges that hold 1.5-ml microcentrifuge tubes, microwell plates and 15- and 50-ml conical tubes
• Chemical fume hood
• Multichannel pipettes and tips
• FloMi filter, 40 μm (VWR, cat. no. 10032-802)
• Falcon cell strainer, 40 μm (VWR, cat. no. 21008-949)
• Pestle for cell strainer (Midsci, cat. no. SG-PEST)
• 96-well plates (Eppendorf, cat. no. 951020401)
• 96-well LoBind plates (Eppendorf, cat. no. 30129512) or FrameStar 96-well skirted PCR plates (Thomas Scientific, cat. no. 1149V59) if LoBind plates are not available
• Thermomixer
• Sonicator (Diagenode Bioruptor Plus)
• Cell counter with GFP channel, or a hemocytometer and an inverted microscope that allows visualization with GFP
• Electrophoresis chambers for PAGE and agarose gels

Buffers and Solutions

• 10× PBS-hypotonic stock solution
  Mix 5.45 g of Na2HPO4 (dibasic), 3.1 g of NaH2PO4·H20, 1.2 g of KH2PO4, 1 g of KCl and 3 g of NaCl in nuclease-free water and bring to a final volume of 500 ml. This stock solution will have a pH of ~6.8, but when diluted to 1×, should end up at pH 7.0–7.4. The buffer can be stored at room temperature (20–23 °C) for 6 months.
• Hypotonic lysis buffer solution A
  This buffer is used for whole mouse embryos E16.5 and older. Mix 5 ml of the 10× PBS-hypotonic stock solution, 5.7 g of sucrose, 75 μl of 2 M MgCl2 and nuclease-free water to a final volume of 50 ml to make the lysis base solution. Right before lysis, for every 1 ml of lysis buffer needed, add 2.5 μl of 10% (vol/vol) IGEPAL (vol/vol) and 10 μl of DEPC and then vortex the solution to disperse the DEPC throughout. For example, if a sample needs 5 ml of lysis buffer, take a 5-ml aliquot of lysis buffer stock solution and add 12.5 μl of 10% (vol/vol) IGEPAL and 50 μl of DEPC. Keep the buffer on ice. Make fresh for each experiment.

• Hypotonic lysis buffer solution B
  This buffer is used for Tiny-Sci, cell lines, mouse embryos under E16.5 and isolated tissues. Mix 5 ml of the 10× PBS-hypotonic stock solution, 75 μl of 2 M MgCl2 and nuclease-free water to a final volume of 50 ml to make the lysis base solution. Right before lysis, for every 1 ml of lysis buffer needed, add 40 μl of BSA (20 mg/ml), 2.5 μl of 10% (vol/vol) IGEPAL and 10 μl of DEPC and then vortex the solution to disperse the DEPC throughout. For example, if a sample needs 5 ml of lysis buffer, take a 5-ml aliquot of lysis buffer base solution and add 200 μl of BSA, 12.5 μl of 10% (vol/vol) IGEPAL and 50μl of DEPC. Keep the buffer on ice. Make fresh for each experiment.

• 0.3 M SPBSTM (sucrose PBS TritonX MgCl2)
  This is the main buffer used throughout the protocol for washing and diluting nuclei. Dissolve 28.5 g of sucrose in 25 ml of 10× DPBS (regular DPBS, not the hypotonic version) and 125 ml of nuclease- free water (about half the volume of water that you will need). Once the sucrose has dissolved, add 2.5 ml of 10% (vol/vol) Triton X-100, 375 μl of 2 M MgCl2 and more water to the final volume of 250 ml. Store this buffer at 4 °C for ≤3 months.
• DSP 50 mg/ml stock
  

Dissolve a 50-mg vial of DSP in 1 ml of anhydrous DMSO (use a new vial of DMSO), because DSP will precipitate in aqueous solutions. Dissolved DSP should be used immediately for practical reasons, this is not necessary. Store the stock solution at -80 °C for \lt 3 months.

• Yoyo-1 dye for counting
  Dilute 1 μl of Yoyo-1 dye in 1 ml of 0.3 M SPBSTM in a dark or amber microcentrifuge tube and store the reagent at 4 °C for ≤3 months. This will be used to dilute nuclei for counting.
• Annealed N7 oligos
  The annealed N7 oligos are Tn5-N7 (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3') and ME (5'-[phos]CTGTCTCTTATACACATCT-3'). Resuspend both oligos to 100 μM in annealing buffer (50 mM NaCl, 40 mM Tris-HCl pH 8.0). Mix one volume of Tn5-N7 with one volume of ME. This creates a working stock at 50 μM. Anneal them with the following PCR program: 95 °C for 5 min, cool to 65 °C (0.1 °C/s), 65 °C for 5 min, cool to 4 °C (0.1 °C/s). Store annealed oligos at 4 °C for 6 months or divide them into aliquots and freeze them at 20 °C for ≤1 year.
• N7-loaded Tn5
  To 20 μl of Tn5, add 20 μl of annealed N7 oligos. Place in a thermomixer and shake at 350 rpm and 23 °C for 30 min. Add 20 μl of glycerol. Store at -20 °C for ≤6 months.
• Tagment DNA buffer (2×)
  To 38.75 ml of nuclease-free water, add 1ml of 1M Tris pH7.6, 250μl of 2M MgCl₂ and 10 ml of dimethylformamide. The final volume is 50 ml. Make 550 μl aliquots and store them at -20 °C for ≤6 months.
• Indexed primer plates
  Primers for reverse transcription, ligation and PCR indexing steps are ordered at 100 μM. Working dilutions are made to 10 μM in EB (10 mM Tris-Cl pH 8.5) and kept at 4 °C for ≤6 months.

• 10% (vol/vol) IGEPAL
  Dilute 5 ml of IGEPAL in 45 ml of nuclease-free water. Store at room temperature for ≤6 months.
• 10% (vol/vol) Triton X-100
  Dilute 5 ml of Triton X-100 in 45 ml of nuclease-free water. Store at room temperature for ≤6 months.
• 10% (vol/vol) Tween 20
  Dilute 5 ml of Tween 20 in 45 ml of nuclease-free water. Store at room temperature for ≤6 months.
• Protease
  Add 7 ml of water to a bottle of lyophilized Qiagen protease (Qiagen, cat. no. 19157). Make 200-μl aliquots and store them at -20 °C for ≤6 months. Do not freeze–thaw.