GSM8280013: Amplicon sequencing of genomic DNA, Immune cell depletion of CD115+ macrophages, batch1, mouse 4, anterior back skin; Mus musculus; OTHER
P60 back skin was harvested with surgical scissors and placed dermis-facing up on a styrofoam tray with pins. Fat and subcutis were scraped off with a scalpel. Fat-free skin was then washed, dermis-side facing down in 1xPBS. Skin was then placed in the same orientation in 0.5% Trypsin-EDTA (10X, Gibco; 15400054) and incubated at 37°C on an orbital shaker for 25 (females) or 50 min (males). Using a glass microscopy slide, skin was secured to the bottom of the dish and scraped again with a scalpel until it started to break. Excess cold PBS with 2% chelexed FBS (FBS(-)) was added to neutralize trypsin. Cell suspension was strained on ice first through a 70 µm filter, washed with an additional 15 mL of cold 1X PBS with 2% FBS(-) and then filtered through a 40 µm filter. Cell suspension was spun down at 400 x g for 10 min. Genomic DNA was extracted with Qiagen DNEasy genomic DNA isolation 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 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.