title: GSM9161534: Osimertinib single compound treatment replicate 2; Mus musculus; OTHER
library_construction_protocol: Genomic DNA (gDNA) was extracted from ~1.5 × 10⁷ cells using salt precipitation56. Cells were resuspended in 200 µL of DPBS in a 15 mL tube. Lysis was achieved by adding 3 mL of lysis buffer (10 mM Tris, 100 mM EDTA, 2% SDS, pH 8.0) containing RNase A (10 mg/mL), followed by vortexing and incubation at 37°C for 5 minutes, then cooling on ice for 3 minutes. Protein contaminants were removed by adding 1 mL of protein precipitation solution (7.5 M ammonium acetate), vortexing, and centrifuging at 2,000 × g for 10 minutes. The supernatant was transferred to a new tube containing 3 mL of isopropanol to precipitate DNA, and the mixture was gently inverted 50 times. DNA was pelleted by centrifugation at 2,000 × g for 3 minutes, washed with 3 mL of 70% ethanol, and air-dried for 5–10 minutes. The pellet was resuspended in 400 µL of hydration buffer (10 mM Tris, pH 8.0), incubated at 65°C for 1 hour, and hydrated overnight at room temperature with shaking at 600 rpm. cDNA were sequenced using a two-step barcoded subamplicon PCR strategy. In the first step, the EGFR kianse domain was divided into two equal subamplicons, and each half was amplifed in a separate reeation. Approximately 5 µg of the DMS library DNA was split between these two reactions, each containing primers encoding 8 degenerate nucleotides to introduce unique molecular identifiers (UMIs). Following amplification, PCR1 products were purified by isopropanol precipitation to remove residual primers and short fragments, and the concentration of each purified product was determined using the PicoGreen method (Invitrogen #P11496). For the second PCR step (PCR2), 500 fg of the purified PCR1 product was used as the template for each subamplicon. This PCR step incorporated Illumina sequencing adapters and sample-specific indexes for multiplexed sequencing. The resulting indexed libraries were then purified using SPRI beads. Libraries were pooled at equimolar concentrations and sequenced on an Illumina NovaSeq SP (500 cycles) platform using paired end reads configured to capture the UMI, barcode, and subamplicon regions.