Publication protocol
Library preparation: AmpliSeq technology is an ultra-high multiplex PCR method that utilizes up to 6144 PCR primer pairs in one tube (Yousem et al., 2013). Two primer pools were used for AmpliSeq target enrichment. For cell identification, the AmpliSeq™ SNP HID panel (Life Technologies) was used which interrogated 136 SNPs of high discriminatory power with a genotype concordance probability of 1031–35 (Pakstis et al., 2010, Sanchez et al., 2006). Although a 340 SNP panel was available for this technology, this panel provided sufficient discriminatory power and was cost effective.
For cancer gene profiling, we used AmpliSeq Cancer hotspot panel version 2 (Life Technologies), which included 207 primer pairs per tube to detect 50 cancer gene hotspots. DNA was extracted from a bulk population of A549 cells (≈ 106 cells), and 10 ng of DNA (≈ 3000 genome copies) was used as a PCR template. Amplicons were generated in a single PCR reaction tube with an endpoint thermal cycler. A total of 50 ng (single cell Library prep replicate #1) and 10 ng (single cell Library prep replicate #2) of WGA-amplified DNA from a single cell were subjected to PCR using the same conditions as above. The amplicons were partially digested and phosphorylated according to the manufacturer's instructions. Amplicons were ligated to adapters included in an Ion Xpress™ Barcode Adapters 1-16 kit (Life Technologies), nick-translated, and then subjected to another round of PCR to complete the linkage between adapters and amplicons. A BioAnalyzer High Sensitivity DNA kit (Agilent Technologies) was used to visualize the size range and determine the library concentration.
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