GSM7500349: Sample_12_guides; 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 and resuspended in FACS buffer before sorting mCherry positive cells on a BD Aria II. The sgRNA containing region has been amplified from BD Rhapsody beads in a separate nested PCR reaction. In PCR1, we used 100 µl KAPA HiFi HotStart ReadyMix (Roche; 07958935001), 6 µl forward primer (5? ACACGACGCTCTTCCGATCT 3?, 10 ?M), 6 µl reverse primer (5?TCTTGTGGAAAGGACGA 3? 10 ?M), 12 ?l Bead RT/PCR Enhancer reagent from BD Biosciences and 72 ?l nuclease free water for a total volume of 200 ?l. Rhapsody bead from each separate cartridge preparation were resuspended in PCR mix and aliquoted in 4x 50 ?l reactions and quickly moved into a pre-heated PCR machine without allowing beads to settle with the following conditions: Initial denaturation at 95°C for 5 minutes, followed by 25 cycles of denaturation 95°C - 30s, annealing 53°C - 30 s, extension 72°C - 20 s, and a final extension of 10 min at 72°C. PCR1 product was pooled, beads were removed magnetically and amplicons were cleaned up with Agencourt AMPure XP beads (Beckman Coulter; A63881) according to manufacturer?s instructions before the second PCR. For PCR 2, 3 ?l of cleaned up PCR1 was used as template along with 2 ?l forward primer (5? ACACGACGCTCTTCCGATCT 3?, 10 ?M), 2 ?l reverse primer (CAGACGTGTGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA*C*C*G 3?, 10 ?M), 18 ?l nuclease free-water, along with 25 ?l of KAPA HiFi HotStart ReadyMix for a total volume of 50 ?l. PCR2 was performed under following conditions: Initial denaturation at 95°C for 3 minutes, followed by 10 cycles of denaturation 95°C - 30 s, annealing 60°C - 3 min, extension 72°C - 60 s, and a final extension of 5 min at 72°C. PCR2 product was cleaned up with Agencourt AMPure XP beads according to manufacturer?s instructions before the indexing PCR. Indexing PCR was performed according to BD Biosciences ?mRNA Targeted Library Preparation? protocol(Doc ID: 210968 Rev. 3.0).