NextSeq 550 paired end sequencing; Evaluation of Tn5 protein fusions suitable for GET-seq
Evaluation of Tn5 protein fusions suitable for GET-seq
Cells were kept in culture according to standard tissue culture procedure. Cells were detached from plates by trypsin treatment, harvested and counted by Trypan Blue exclusion. Nucleic acids were directly processed on permeabilized nuclei following standard ATAC-seq protocol  (https://doi.org/10.1038/nmeth.2688) with minor modifications. Briefly, 100,000 Caki-1 cells pellets were washed in 100 μL cold 1X PBS, centrifuged for 10 min at 500 *g at 4 °C, and permeabilized in 100 μL of cold lysis buffer (10 mM Tris·Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) Igepal CA-630), then centrifuged again for 10 min at 500 *g at 4 °C. Nuclei were resuspended in  100 μL of transposition mix (5x TAPS-PEG8000 buffer,10 μL of 1.39 μM of functional TnH or Tn5 transposome, water to in a final volume of 100 μL). As control, a parallel reaction was carried out on 100,000 Caki-1 cells pellets using the TDE Nextera transposase (Illumina) according to the manufacturer's protocol (50 μL 2× TD buffer, 5 μL TDE and 45 μL nuclease-free water). Reactions were performed at 37 °C for 30 min,  stopped adding SDS at a final concentration of 0.05% and incubated for 5 min at RT. Reactions were purified using QIAquick Gel Extraction Kit (Qiagen) and eluted in 15 μL of EB buffer. 5 μL of this reaction was used as input for indexing PCR using 2X Kapa HiFi polymerase (Roche) and Nextera indexing primers (N7XX and S5XX). Libraries clean up was performed following standard procedure.