SAMOSA-Tag preparation of prostate metastasis PDX sample, unmethylated.
Nuclei from a tumor sample are treated with 1 mM SAM for 30 min at 37ºC. Then, nuclei are directly tagmented using custom-loaded Tn5 transposase with hairpin adapters compatible with PacBio SMRT sequencing in Omni-ATAC Buffer (10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.33X PBS, 10% DMF, 0.01% digitonin, 0.1% Tween-20) for 45 - 60min at 55ºC. Nuclei are then treated with 10 µg RNaseA at 37ºC for 15 min, and lysed by addition of 50 µg Proteinase K in 2.5 µL of 10% SDS and 2.5 µL of 0.5 M EDTA at 60ºC with 1000 RPM shaking for 1-2 h. Resulting fragments are cleaned up using a 2X SPRI and gap-repaired using a polymerase / ligase combination (Phusion polymerase / Taq ligase) at 37ºC for 1hr. Libraries are then digested with Exonuclease III at 37ºC for 1 hr to remove un-repaired, non-productive templates.