Nextera tde1

Approximately 5 × 10⁴ pooled fresh HCAMSC cells were collected by centrifugation at 500 rcf and washed twice with cold PBS. Nucleus-enriched fractions were extracted with cold resuspension buffer (0.1% NP-40, 0.1% Tween-20, and 0.01% Digitonin) and washed out with 1 mL of cold resuspension buffer containing 0.1% Tween-20 only. Nucleus pellets were collected by centrifugation and resuspended with transposition reaction buffer containing Tn5 transposases (Illumina Nextera TDE1). Transposition reactions were incubated at 37 °C for 30 min, followed by DNA purification using the DNA Clean-up and Concentration kit (Zymo D4013). Libraries were amplified using Nextera barcodes and High-Fidelity polymerase (NEB M0541S) and purified using Agencourt Ampure XP beads (Beckman Coulter A63880) double-size selection (0.5X:0.9X). For qPCR experiments, transposed samples were normalized by genomic DNA which was extracted using Quick-DNA Microprep Kit (Zymo D3020). Library was sequenced on the Illumina HiSeq X instrument to 400 million 150-bp paired-end reads.

Five million fungal spheroplasts or 0.5 ng naked gDNA were resuspended in the tagmentation reaction mix (12.50 μL Nextera 2× TD buffer, 2.00 µL Nextera TDE1 (all Illumina), 0.50 µL 50× protease inhibitor cocktail (Roche, Basel, Switzerland), 0.25 µL 1% Digitonin (New England Biolabs, Ipswich, MA, USA, Frankfurt am Main, Germany) and 10.25 µL nuclease-free distilled H₂O (ThermoFisher Scientific, Shanghai, China) and incubated at 37 °C for 30 min. The tagmentation reaction was immediately purified using a Qiagen MiniElute PCR purification kit and elution was done in 12 µL elution buffer provided by the kit.