Herculase 2 enzyme

Large scale parts synthesis was performed by PCR in 96-well plates using sequence verified master plasmid templates. Each reaction contained 1 ng of plasmid template, 1x Herculase II reaction buffer (Agilent Technologies, Inc.), 0.25 mM of each dNTP, 0.4 μM of each PCR primer and 2 μl of pre-formulated Herculase II enzyme (Agilent Technologies, Inc.) in a final volume of 100 μL. Thermocycling conditions were: 1 cycle at 95°C for 2 min.; 30 cycles at 95°C for 20 sec., 55°C for 20 sec. and 72°C for 30 sec.; 1 cycle at 72°C for 3 min. Contents of multiple 96-well plates for each SV part were pooled and purified using AMPure XP magnetic beads according to the manufacturer’s instructions (Beckman-Coulter). Correct lengths and purities of SV parts were assessed with an Agilent Technologies, Inc. BioAnalyzer.

Investigation of CD19 cDNA of individual cells was performed by nested-PCRs using amplified 10× barcoded full-length cDNA. Three successive PCR were carried out with Herculase II enzyme (Agilent technologies) and with the following PCR program: 95 °C 1 min, 20 cycles (95 °C 20 s, 56 °C 20 s, and 68 °C 90 s), 68 °C 4 min (for the third PCR only 15 cycles were performed). In the first PCR, CD19 transcript was amplified with “partial Read1” primer (or P12) and CD19 primer P10. Cell-specific CD19 cDNA amplifications were carried out with primers comprising the 10× cell BC (BC primers) and P14 or P13 for respectively second and third PCR (Supplementary Table 1). For these nested-PCR, 1 µl aliquots of primary or secondary PCR were used. PCR products were analyzed by 1% agarose gel electrophoresis.