Droplet generator

cDNA was synthesized in the same methods described in the Quantitative RT-PCR section.
cDNA and EML4-ALK specific probes ddPCR EXD Assay EML4-ALK (Bio-Rad Laboratories, dHsaEXD86850342, Hercules, CA, USA) were mixed with ddPCR Supermix for Probe (no dUTP) (Bio-Rad Laboratories). Droplet was generated from the cDNA and probe mixture using Droplet Generator (Bio-Rad Laboratories) according to the manufacturer’s instructions. PCR was performed using C1000 Touch thermal cycler (Bio-Rad Laboratories) with the cycle described in the manufacturer’s protocol of the probes. After PCR, measurement of droplet was performed using Droplet Reader (Bio-Rad Laboratories) and QuantaSoft Analysis Pro Software (Bio-Rad Laboratories) was used for analysis.

The QX200 droplet digital PCR (ddPCR) system (Bio-Rad) was used to measure gene expression levels in iPSCs and iPSC-derived neurons. The reaction was carried out in 20 μl emulsion PCR reactions that contain 20,000 droplets. Total RNAs were extracted by RNeasy Mini kit (Qiagen, 74104) and genomic DNA was removed by column by RNase-Free DNase (Qiagen, 79254). The ddPCR assay consisted of the following components: 1 × One-Step RT-ddPCR mix for probes (Bio-Rad, 1864021), forward primer (900 nM), reverse primer (900 nM), probe (FAM or HEX, 250 nM), nuclease-free water, and 5 ng RNA. All primers and probes were purchased from Thermo Fisher Scientific (MAP2, Hs00258900; OCT4, Hs04260367; BRN2, Hs00271595; FOXG1, Hs01850784; SYN1, Hs00199577) or Bio-Rad (RPP30, 10031228). Droplets were generated in a droplet generator (Bio-Rad) and PCR was performed in a C1000 Touch thermal cycler (Bio-Rad) according to the manufacturer's recommendation. After PCR, readout of positive versus negative droplets was performed using a QX200 droplet reader (Bio-Rad) and calculated by QuantaSoft software version 1.7.4.0917 (Bio-Rad).

The translocation assay targets PCSK9 and HBEGF and was performed as described previously^{86 (link)}. In short, genomic DNA was isolated from HEK293T cells 3 days after transfections using the Gentra Puregene Cell Kit (Qiagen) and was diluted to 10 ng/μL. Custom ddPCR assays were ordered from Bio-Rad detecting balanced translocations between PCSK9 and HBEGF (Supplementary Data 2). AP3B1 (BioRAD) was used as reference assay. ddPCR PCR reaction contained 1× ddPCR Supermix for Probes, no dUPT (Bio-Rad), 1× FAM-labelled HBEGF-PCSK9 custom assay (BioRAD), 1× AP3B1-HEX labelled human reference assay (Bio-Rad), 1/40 HaeIII (Invitrogen) and 50 ng/μL genomic DNA. 20 µL PCR reaction was used to generate lipid droplets with an automated Droplet Generator (Bio-Rad). PCR amplification was performed using the following conditions: 95 °C for 10 min, 40x (94 °C for 30 s, ramp 2 °C/s; 63.2 °C for 1 min) followed by enzyme deactivation at 98 °C for 10 min. Readout was performed with QX 100 Droplet Reader (Bio-Rad) and ddPCR Droplet Reader Oil (Bio-Rad). Data analysis was conducted with QuantaSoft 1.7.4 Software from Bio-Rad.

Next, CFP10 and Rv1768 DNA copy numbers were quantified in the same samples using the QX100™ Droplet Digital™ PCR system (Bio-Rad, CA, USA). The 20 μl ddPCR mixture comprised of 10 μl of 2 × ddPCR Supermix (Bio-Rad, CA, USA), 1.0 μl of 10 μM sense and antisense primer, 0.5 μl of 10 μM probe, 2 μl of 100 ng/μl whole-blood genomic DNA or plasmid DNA, and RNase/DNase-free water to a final volume of 20 μl. The mixture was placed into the DG8 cartridge with 70 μl of droplet generation oil (Bio-Rad, CA, USA), and the droplets were formed in the droplet generator (Bio-Rad, CA, USA). Subsequently, the droplets were transferred to a 96-well PCR plate (Eppendorf, Hamburg, Germany), and PCR amplification was performed on a S1000 thermalcycler (Bio-Rad, CA, USA) using the following parameters: 95 °C for 5 min, followed by 40 cycles at 95 °C for 30 s and 60 °C for 60 s, with a final hold for 10 min at 98 °C. After amplification, the plate was loaded on the droplet reader (Bio-Rad, CA, USA), and the droplets from each well of the plate were automatically read at a rate of 32-wells per hour. The ddPCR data were analyzed using QuantaSoft analysis software (Bio-Rad, CA, USA), and the quantification of the target genes is presented as the number of copies per μl in PCR mixture. The recombinant plasmids were used as templates to draw the standard curves.

For mutation detection, the QX100 ddPCR System (Bio‐Rad Laboratories, Hercules, CA, USA) along with primers and probes (FAM, mutant‐type, and HEX, wild‐type) and ddPCR Supermix for probes (No dUTP) from Bio‐Rad Laboratories were employed according to the manufacturer's protocol. Details on the primers and probes for TERT promoter mutants C228T and C250T used in the ddPCR analyses are shown in Table S1. Droplets were generated using a droplet generator (Bio‐Rad Laboratories). The PCR cycling parameters included 10 min at 95 °C, 50 cycles of 94 °C for 30 s and 55 °C for 1 min, and one cycle of 98 °C for 10 min followed by a 12 °C hold. Droplet fluorescence was assessed using a droplet reader. Analysis of the ddPCR data for allele calling and the calculation of absolute copy numbers were performed using quantasoft software, version 1.7.4 (Bio‐Rad Laboratories). The samples were considered true positive for targeted mutations when the following two criteria were met. First, they contained at least three droplets in the positive area of the FAM signal. Second, the mutant allele frequency (MAF) was > 5.0% to exclude false‐positive reactions induced by DNA degradation due to the fixation process. MAF was defined as the proportion of mutant‐type copies relative to the total number of copies including both mutant‐type and wild‐type alleles as determined by the ddPCR analyses.