title: GSM8742671: loxP6 design, clonal, Cas9 sequencing, bright gate, replicate 2; Homo sapiens; OTHER
library_construction_protocol: Between 2 and 4 million cells were collected by centrifugation, and high molecular weight (HMW) genomic DNA was extracted using the Monarch High Molecular Weight DNA Extraction Kit (New England BioLabs) with agitation speeds set to 1,500 rpm or 2,000 rpm. A modified version of the Monarch protocol was used to simultaneously assess DNA accessibility when required. In this modified protocol, 129 µl of nuclei prep buffer was combined with 10 µl of recombinant Cutsmart, 1 µl of 32 mM S-adenosylmethionine (final concentration 160 µM), 10 µl of 5000 Units/ml EcoGII methyltransferase (50 Units; New England BioLabs), and 5 µl of 20 mg/ml RNase A (200 µg; New England BioLabs). Approximately 2–3 million cells were resuspended in this reaction buffer and incubated at 37°C for 30 minutes. The reaction was then heated to 65°C for 5 minutes to inactivate the enzymes. Subsequently, 150 µl of nuclei prep buffer containing 10 µl of proteinase K was added to the nuclei preparation, and HMW DNA was extracted following the standard Monarch High Molecular Weight DNA Kit protocol. For samples containing six loxPsym sites obtained from screens, an additional step was included to eliminate low-molecular-weight DNA. This was achieved using the Short Fragment Eliminator Kit (Oxford Nanopore Technologies), following the manufacturer's instructions. Cas9-targeted sequencing: Cas9 enrichment was carried out using the Cas9 sequencing kit protocol with minor modifications (CAS9106 Protocol v109, Oxford Nanopore Technologies). Briefly, 5 µg of high molecular weight (HMW) DNA was dephosphorylated. tracrRNA and crRNA were annealed and complexed with HifiCas9 V3 (Integrated DNA Technologies) to form RNPs. The dephosphorylated DNA was incubated with Cas9 RNPs for 30 minutes at 37°C. Sequencing adapters were then ligated to the cleaved DNA, with the ligation step extended to 1 hour at room temperature. The libraries were purified using Ampure XP beads (Agilent), washed with Long Fragment Buffer, and eluted in an elution buffer for 16 hours. Sequencing was performed using the MinION Mk1B platform with R9.4.1 flow cells (FLO-MIN106). Long-range PCR: PCR amplification was performed using the LongAmp Hot Start Taq 2X Master Mix (New England BioLabs), using 250 ng of genomic DNA as input and 1 µM of forward and reverse primers, under the following cycling conditions: initial denaturation at 94°C for 30 seconds, followed by 30 cycles of 94°C for 30 seconds, 59°C for 1 minute, and 65°C for 25 minutes, with a final extension at 65°C for 10 minutes. PCR products were purified using AMPure XP Beads (Beckman Coulter) at a bead-to-sample ratio of 0.66X and eluted with the elution buffer from the Monarch HMW DNA Extraction Kit for Cells & Blood. Genomic DNA from cell samples sorted into different gates was amplified using primers with custom index extensions for multiplexing. The resulting amplicons were sequenced on the MinION Mk1B platform using R10.4.1 flow cells (FLO-MIN114).