GSM8280129: Amplicon sequencing of genomic DNA, DMBA/TPA-induced tumors, replicate 79; Mus musculus; OTHER
DMBA/TPA-induced tumors (which were not used for neither scRNA-seq nor spatial analysis) were minced and genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit. The sgRNA region was amplified in a nested PCR. In PCR1, we used 12.5 µl KAPA HiFi HotStart ReadyMix (Roche, 07958935001), 0.75 µl forward primer (5' CTTGTGGAAAGGACGAAACACCG 3', 10μM), 0.75 µl reverse primer (5' GTGTCTCAAGATCTAGTTACGCCAAGC 3' 10μM), 1 µl of extracted genomic tumor DNA (with a concentration between 50-100 ng/μl) and 10 μl nuclease-free water for a total volume of 25 μl per tumor. PCR1 was performed using the following conditions: Initial denaturation at 98°C for 2:30 min, followed by 22 cycles of denaturation 98°C -– 20 s, annealing 62°C -– 30 s, extension 72°C -– 30 s. PCR1 products for each tumor were individually column-purified with a GenElute™ PCR Clean-Up Kit (Sigma-Aldrich NA1020-1KT). For PCR2, 1 μl of cleaned up PCR1 was used as template along with 0.75 μl forward barcoded i5 primer (see Supplementary Information Table 1, 10 μM), 0.75 μl reverse barcoded i7 primer (see table, 10 μM), 10 μl nuclease free-water, along with 12.5 μl of KAPA HiFi HotStart ReadyMix for a total volume of 25 μl. Eight different i5 and i7 primers allowed for a total of 64 different combinations. PCR2 was performed under following conditions: Initial denaturation at 95°C for 3 min, followed by 32 cycles of denaturation 98°C -– 30 s, annealing 62°C – 30 s, extension 72°C – 30 s. PCR2 products for a maximum of 64 individually barcoded tumor amplifications were pooled and cleaned up with Agencourt AMPure XP beads (Beckman Coulter, A63881) according to manufacturer's instructions before sequencing.