title: In situ sequencing of early mouse embryos
design: Following in situ sequencing of polyacrylamide-embedded embryos, the gels were washed with ultra pure water, scored with a razor, scraped off of the glass-bottom plate in pieces using a disposable polypropylene spatula, and transferred using a paintbrush (Blick Art Supplies, 06170-7030) to PCR tubes containing 34 ul of PCR mix each. PCR mix was prepared as follows: 20 L Phusion U Hot Start PCR Master Mix, 1 L 20 M P5 primer, 2 L 10 M i7 indexed Nextera primer (Illumina FC-131-1002), 11 L ultra pure H2O. The gel fragments were assumed to contain 6 L of ultra pure water. The tubes were flicked several times, then incubated on ice 3 times for 10 minutes with flicking between each incubation. The tubes were then pre-amplified by thermocycling according to the following program: 98C for 30 s, 5 cycles of [98C for 45 s, 72C for 80 s], 4C hold. The tubes were then flicked and incubated at 4C overnight to allow amplicons to diffuse out of the gel. The tubes were flicked again. 1.5 L of the pre-amplified libraries was diluted 1:10 in PCR mix with 1x SYBR Green followed by quantification by qPCR. Half the remaining volume of the pre-amplified libraries was then diluted 1:10 in PCR mix and thermocycled according to the following program: for cycles for each diluted library (ranging from 18-24 cycles): 98C for 30 s, [# qPCR cycles required to reach -maximum + 1] cycles of [98C for 10 s, 72C for 40 s], 72C for 5 min, 4C hold. The number of cycles used in the latter PCR reaction ranged from 18-24. Dilution of the pre-amplified libraries was to dilute putative carry-over from the in situ sequencing, which dramatically improved yield.