title: In situ genome sequencing of human fibroblasts
design: Following in situ sequencing of cultured cells, the coverslip was removed from the flow cell. The coverslip was cut down to the size of the imaged region (~250 cells) using a diamond scribe, and placed the remaining fragment in a PCR tube. The coverslip was cut to fit the sample in a PCR tube. A conventional Illumina sequencing library was prepared from the sample as follows: the trimmed coverslip fragment was fully immersed by adding 100 L PCR mix (50 L Phusion U Hot Start PCR Master Mix (Thermo F533), 0.5 L of 100 uM P5 primer, 0.5 L of 100 M v2_Ad2.41 primer, 49 L ultrapure water), and performed a first round of amplification by incubation at 8C for 10 min, 98C for 30 s, 10 cycles of [98C for 10 s, 69C for 15 s, 72C for 30 s], 72C for 5 min, 4C hold. To avoid overamplification, 5 L was eluted for quantification by qPCR, as described in (8), maintaining the rest of the sample at 4 C. An additional 15 cycles of amplification was performed on the remaining 95 L of sample as described above. Following PCR, the reaction was eluted from the PCR tube, column purified (DNA Clean and Concentrator-5, Zymo) using 475 L DNA binding buffer (i.e. 1:5) and eluted into 20 L water.