All cell sorting was performed on the BD Influx™ cell sorter using BD FACS™ Sortware software. The standard plate holder apparatus for the Influx was filed down to accommodate a 96-well cooling block (BioCision CoolRack®, San Rafael, CA), which could hold BD Precise™ Single Cell Encoding 96-well plates in a rigid position to enable sorting.
For WTA analysis, YFP positive cells were sorted into chilled 0.2 ml PCR tubes (Applied Biosystems, Foster City, CA) containing 5 μl of PBS (w/o Ca/Mg) and SUPERaseIn™ RNase inhibitor (Invitrogen, Carlsbad, CA) at 1 U/ml. The tubes were centrifuged at 300 g for 10 minutes followed by addition of 5 μl of 2x Single-Cell Lysis Buffer (Takara Bio USA, Inc., Mountain View, CA) with SUPERaseIn™ RNase inhibitor at 2 U/ml. The lysates were stored at −80 °C until cDNA synthesis.
For Precise workflow, cells were sorted directly into chilled BD Precise™ Single Cell Encoding 96-well plates containing lysis buffer, indexing dT primers and dNTPs. After sorting, plates were sealed with a foil cover, vortexed for 5–10 seconds, briefly centrifuged and frozen at −80 °C until further analysis.
For WTA analysis, YFP positive cells were sorted into chilled 0.2 ml PCR tubes (Applied Biosystems, Foster City, CA) containing 5 μl of PBS (w/o Ca/Mg) and SUPERaseIn™ RNase inhibitor (Invitrogen, Carlsbad, CA) at 1 U/ml. The tubes were centrifuged at 300 g for 10 minutes followed by addition of 5 μl of 2x Single-Cell Lysis Buffer (Takara Bio USA, Inc., Mountain View, CA) with SUPERaseIn™ RNase inhibitor at 2 U/ml. The lysates were stored at −80 °C until cDNA synthesis.
For Precise workflow, cells were sorted directly into chilled BD Precise™ Single Cell Encoding 96-well plates containing lysis buffer, indexing dT primers and dNTPs. After sorting, plates were sealed with a foil cover, vortexed for 5–10 seconds, briefly centrifuged and frozen at −80 °C until further analysis.