Single-Cell RNA Sequencing of Circulating Tumor Cells

Single CTCs were recovered, using the nanowell-based isolation platform, into individual wells of a 96 well plate containing 10 µL of buffer TCL (Qiagen) supplemented with 1% 2-mercaptoethanol (Sigma), spun down, snap frozen on dry ice, and stored at −80 °C until further processing. Next, RNA from each single CTC was isolated, reverse transcribed, and amplified using the SMARTer Ultra-low RNA kit (Clontech) as previously described^{32 (link)}. Afterwards, cDNA libraries were prepared using Nextera XT DNA Sample preparation reagents (Illumina) as per the manufacturer’s recommendations, with minor modifications. Specifically, reactions were run at ¼ the recommended volume, the tagmentation step was extended to 10 minutes, and the extension time during the PCR step was increased from 30s to 60s. After the PCR step, all 96 samples were pooled without library normalization, cleaned twice with 0.9× AMPure XP SPRI beads (Beckman Coulter), and eluted in Tris-EDTA buffer, pH 8 (Teknova). The pooled libraries were quantified using Quant-IT DNA High-Sensitivity Assay Kit (Invitrogen) and examined using a high sensitivity DNA chip (Agilent). Finally, samples were sequenced using a MiSeq sequencer (Illumina).