title: GESTALT barcode sequencing of Danio rerio organ: Brain.Fish5
design: GESTALT cells were transplanted to wild-type host embryos; embryos grown to adults; organs isolated and barcodes were PCR amplified from this organ. Two primers flanking the barcode were used in an initial PCR reaction; samples from fish 1-10 used a different set of primers than samples from fish 11-20. After 0.9X AMPure cleanup of the initial PCR amplicon, two additional primers added indexes and adapters in a second PCR reaction. Each indexing primer in the second PCR was used twice on two distinct samples; thus 40 indexes were used on 80 samples. A second 0.9X AMPure cleanup preceded roughly equimolar pooling to form a single multiplexed library. Demultiplexing was done first on the indexes and then on the initial PCR primers to demultiplex the 80 samples. Primers for samples from fish 1-10: TCGAGCTCAAGCTTCGG, CTGCCATTTGTCTCGAGGTC; primers for samples from fish 11-20: AGATCTCGAGCTCAAGCTTC, GGGCTGCCATTTGTCTCGAG.