A universal deep-learning model for zinc finger design enables transcription factor reprogramming
library variants, surviving colonies were pooled, miniprepped and DNA barcoded for sequencing on an Illumina NextSeq 500. Two microliters of pooled plasmid DNA was used as a template for barcoding in a 25-μl reaction with Taq Polymerase , with the following cycling parameters: 95 °C for 5 min, 20 cycles of and 68 °C for 10 min, and was then held at 4 C.
HEK293T cells were transfected with ZF activators and repressors, and target transcript levels measured by RT–qPCR as follows. Cells were cultured in DMEM supplemented with 10% FBS, 2 mM GlutaMax , 1% penicillin/streptomycin, 1% MEM nonessential amino acids and 2 mM sodium pyruvate. Medium was changed 2 days post transfection and cells harvested for RT–qPCR 3 days post transfection.
One microgram of pure RNA was reverse transcribed using the SuperScript IV First-Strand Synthesis System according to the manufacturer’s instructions. Random hexamers were used as primers. qPCR reactions were established in technical duplicate or triplicate using the equivalent of 25 or 50 ng of reverse-transcribed RNA per reaction and the KAPA SYBR FAST qPCR Master Mix .
RT–qPCR was performed on a LightCycler 480 Instrument II using the cycling program recommended for KAPA SYBR FAST reagent with the LightCycler 480 . Cycle threshold values were calculated using the on-board Absolute Quantification/2, and fold change in expression for a given gene of interest was calculated relative to the appropriate negative control.
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