GSM7500346: Sample_9_guides; Mus musculus; OTHER
P4 back skin was surgically removed and scraped for fat, the back skin was washed once in cold PBS and then placed in dispase (Corning; 354235) for 35 min at 37°C, epidermis facing up, on an orbital shaker. Next, epidermis was separated from dermis with fine forceps, torn into smaller pieces and placed in 4 mL 0.25% Trypsin-EDTA (1X, Gibco; 25200056) for 15 min at 37°C with orbital shaking. Epidermis was washed with cold PBS and pipetted vigorously to achieve a single cell suspension. Suspension was then afterwards filtered through a 70 µm and a 40 µm strainer (Corning; 431750, 431751) consecutively, then centrifuged for 10 min at 1400rpm and resuspended in FACS buffer (PBS + 2 % FBS(-) + DAPI) 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).