Tn5 transposase purification and loading

Tn5 transposase was expressed as an intein chitin-binding domain fusion and purified using an improved version of the method first described by Picelli et al.⁵⁵. T7 Express lysY/I (C3013I, NEB) cells were transformed with the plasmid pTXB1-ecTn5 E54K L372P (#60240, Addgene)⁵⁵. An LB Ampicillin culture was inoculated with three colonies and grown overnight at 37 °C. The starter culture was diluted to an OD of 0.02 with fresh media and shaken at 37 °C until it reached an OD of 0.9. The culture was then immediately chilled on ice to 10 °C and expression was induced by adding 250 μM IPTG (Dioxane Free, CI8280-13, Denville Scientific). The culture was shaken for 4 h at 23 °C after which cells were harvested in 2 L batches by centrifugation, flash frozen in liquid nitrogen and stored at −80 °C. Cell pellets were resuspended in 20 ml of ice-cold lysis buffer (20 mM HEPES 7.2-KOH, 0.8 M NaCl, 1 mM EDTA, 10% Glycerol, 0.2% Triton X-100) with protease inhibitors (Complete, EDTA-free Protease Inhibitor Cocktail Tablets, 11873580001, Roche Diagnostics) and passed three times through a Microfluidizer (lining covered with ice water, Model 110 L, Microfluidics) with a 5 min cool down interval in between each pass. Any remaining sample was purged from the Microfluidizer with an additional 25 ml of ice-cold lysis buffer with protease inhibitors (total lysate volume ~50 ml). Samples were spun down for 20 min in an ultracentrifuge at 125K × g (L-80XP, 45 Ti Rotor, Beckman Coulter) at 4 °C. ~45 ml of supernatant was combined with 115 ml ice-cold lysis buffer with protease inhibitors in a cold beaker (total volume = 160 ml) and stirred at 4 °C. 4.2 ml of 10% neutralized polyethyleneimine-HCl (pH 7.0) was then added dropwise. Samples were spun down again for 20 min in an ultracentrifuge at 125K × g (L-80XP, 45 Ti Rotor, Beckman Coulter) at 4 °C. The pooled supernatant was loaded onto ~10 ml of fresh Chitin resin (S6651L, NEB) in a chromatography column (Econo-Column (1.5 × 15 cm), Flow Adapter: 7380015, Bio-Rad). The column was then washed with 50–100 ml lysis buffer. Cleavage of the fusion protein was initiated by flowing ~20 ml of freshly made elution buffer (20 mM HEPES 7.2-KOH, 0.5 M NaCl, 1 mM EDTA, 10% glycerol, 0.02% Triton X-100, 100 mM DTT) onto the column at a speed of 0.8 ml/min for 25 min. After the column was incubated for 63 h at 4 °C, the protein was recovered from the initial elution volume and a subsequent 30 ml wash with elution buffer. Protein-containing fractions were pooled and diluted 1:1 with buffer [20 mM HEPES 7.2-KOH,1 mM EDTA, 10% glycerol, 0.5 mM TCEP) to reduce the NaCl concentration to 250 mM. For cation exchange, the sample was loaded onto a 1 ml column HiTrap S HP (17115101, GE), washed with Buffer A (10 mM Tris 7.5, 280 mM NaCl, 10% glycerol, 0.5 mM TCEP) and then eluted using a gradient formed using Buffer A and Buffer B (10 mM Tris 7.5, 1 M NaCl, 10% glycerol, 0.5 mM TCEP) (0% Buffer B over 5 column volumes, 0–100% Buffer B over 50 column volumes, 100% Buffer B over 10 column volumes). Next, the protein-containing fractions were combined, concentrated via ultrafiltration to ~1.5 mg/mL and further purified via gel filtration (HiLoad 16/600 Superdex 75 pg column (28989333, GE)) in Buffer GF (100 mM HEPES-KOH at pH 7.2, 0.5 M NaCl, 0.2 mM EDTA, 2 mM DTT, 20% glycerol). The purest Tn5 transposase-containing fractions were pooled and 1 volume 100% glycerol was added to the preparation. Tn5 transposase was stored at −20 °C.

To generate Tn5 transposomes for combinatorial barcoding assisted single-nucleus ATAC-seq, barcoded oligos were first annealed to pMENTs oligos (95 °C for 5 min, cooled to 14 °C at a cooling rate of 0.1 °C/s) separately. Next, 1 µl barcoded transposon (50 µM) was mixed with 7 ul Tn5 (~7 µM). The mixture was incubated on the lab bench at room temperature for 30 min. Finally, T5 and T7 transposomes were mixed in a 1:1 ratio and diluted 1:10 with dilution buffer (50% Glycerol, 50 mM Tris-HCl (pH = 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100, 1 mM DTT). For combinatorial barcoding, we used eight different T5 transposomes and 12 distinct T7 transposomes, which eventually resulted in 96 Tn5 barcode combinations per sample⁷ (Supplementary Table ⁴). Library quality control for single-nucleus ATAC-seq can be found in Supplementary Table ³.