Sample fracturing, lysis and size-selection

Samples were placed in a stainless-steel vial (Biospec 2007) along with a 6.35 mm stainless steel bead (Biospec 11709635ss), and were sealed with a silicone rubber plug cap (Biospec 2008). The vial was placed in liquid nitrogen for >2 minutes, vigorously shaken to dislodge the sample from the vial wall, and quickly transferred to a bead beater (Biospec 112011) and subjected to beating for 10 seconds. PBS was added to the vial and vortexed; clusters in PBS were removed and washed twice with PBS via centrifugation at 15K RPM for 1 minute (Eppendorf 5424). Next, embedded cells were lysed⁴³; clusters were resuspended in 500 μL lysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM ethylenediaminetetraacetic acid [EDTA], 100 mM NaCl) with 75 U/μL lysozyme (Epicentre R1810M) and were incubated at 37 °C for 1 hour. Clusters were then resuspended in 500 μL digestion buffer (30 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.5% Triton X-100, 800 mM guanidine hydrochloride [Sigma-Aldrich G9284]) with 0.1 μg/μL proteinase K (Epicentre MPRK092), and were incubated at 65 °C for 15 minutes. Finally, clusters were incubated at 95 °C for 5 minutes to inactivate proteinase K and washed three times with TET.

Samples were next subjected to size-selection. Clusters were first passed through a 40 μm cell strainer (Fisher 22–363-547) to remove large particulate matter. Next, nylon mesh filters (Component Supply Company, 7 μm: U-CMN-7-A, 15 μm: U-CMN-15-A, 31 μm: U-CMN-31-A) were cut to size using a ½” hole punch and two filter punches were placed in a holder (EMD Millipore SX0001300) for each size. Clusters were passed through the 31 μm filter, 15 μm filter, and 7 μm filter sequentially using a 3 mL syringe (BD 309657); for each filter, clusters were passed through three times, and retained clusters on filters were washed once with TET. Clusters were washed off the 15 μm filter (large, ~30 μm median diameter) and 7 μm (medium, ~20 μm median diameter) or collected from the pass-through from the final 7 μm filter (small, ~7 μm median diameter). The concentration of clusters was quantified by counting on a hemocytometer (INCYTO DHC-N01) and stored at 4 °C in TET for processing within ~2 days.