Ethidium homodimer-1

Adherent cultures were dissociated using 0.05% Trypsin supplemented with 0.02% EDTA or using TrypLE^™ Select (Life Technologies). Following centrifugation and removal of the dissociation medium, cells were resuspended at a concentration of 150–500 cells/μL. This cell suspension was mixed with C₁^™ Cell Suspension Reagent (Fluidigm, Cat # 634833) at the recommended ratio of 3:2 immediately before loading 5 μL of this final mix on the C₁^™ IFC along with 20 μL of freshly prepared staining buffer (2.5 μL ethidium homodimer-1 and 0.625 μL Calcein AM from Life Technology’s LIVE/DEAD^® Viability/Cytotoxicity Kit added to 1.25 mL C₁^™Cell Wash Buffer) in their respective input wells. Images of captured cells were collected Leica DMI 4000B microscope in the brightfield, GFP, and CY3 channels using the Surveyor V7.0.0.9 MT software (Objective Imaging).
Single-cell RNA extraction and mRNA amplification were performed on the C₁^™ Single-Cell Auto Prep Integrated Fluidic Circuit (IFC) following the methods described in the protocol (PN 100–7168, http://www.fluidigm.com/). For experiments where exogenous spike-in controls were used, the spikes were added to the lysis mix at a 20,000-fold dilution. The PCR thermal protocol was adapted from a recent publication that optimized template-switching chemistry for single-cell mRNA Seq^{32 (link)} and is outlined in the C₁^™ Single-Cell Auto Prep System protocol. For the population control experiment, we used reagent formulations and workflows exactly as described in the SMARTer^® Ultra Low RNA Kit user manual (Cat# 634833,1 kit for 10 C₁^™ IFCs), except that the thermal protocol followed the recommendations outlined in the C₁^™ Single-Cell Auto Prep System user guide (PN 100–7168).
For the population control experiment, we sorted 100 K562 cells into 3.5 μL of Clontech Reaction Buffer containing exogenous spike-in controls using a BD FACSAria^™ III. The 20,000-fold diluted ERCC spike-in controls were further diluted (9:3500) in Clontech Reaction Buffer such that an equal mass (rather than an equal concentration) of the spikes was included in the population control reaction. Following the sort, cells were frozen at −80 °C overnight before continuing the SMARTer^® Ultra Low RNA Kit protocol according to manufacturer’s recommendations.
The cDNA reaction products were quantified using the Quant-iT^™ PicoGreen^® dsDNA Assay Kit (Life Technologies) and high sensitivity DNA chips (Agilent) and were then diluted to a final concentration of 0.15–0.30 ng/μL using C₁^™ Harvest Reagent. The diluted cDNA reaction products were then converted into mRNA Seq libraries using the Nextera^® XT DNA Sample Preparation Kit (Illumina, FC-131-1096 and FC-131-1002, 1 kit used for 4 C₁^™ IFCs/384 samples) following manufacturer’s instructions, with minor modifications. Specifically, reactions were run at one quarter of the recommended volume, the tagmentation step was extended to 10 minutes, and the extension time during the PCR step was increased from 30 seconds to 60 seconds. After the PCR step, samples were pooled, cleaned twice with 0.9X Agencourt AMPure XP SPRI beads (Beckman Coulter), eluted in TE buffer and quantified using a high sensitivity DNA chip (Agilent). For high-coverage sequencing, libraries from a subset of captured cells from each source were pooled to reach a target of ten million aligned reads per cell.