title: GSM9282396: tumor_1; Homo sapiens; OTHER
library_construction_protocol: Genomic DNA was extracted from tumors and pre-injected cell pellets using the methods from a previous study. Briefly, each sample was put in a 15 mL Falcon tube and 6 mL NK Lysis Buffer (50 mM Tris, 50 mM EDTA, 1% SDS, pH adjusted to 8.0) along with 30 μL of 20 mg/mL Proteinase K (Qiagen) were added. These samples were then incubated at 55 °C overnight. After tissue digestion, 30 μL of 10 mg/mL RNase A (Qiagen) was added to the lysed sample, which was then inverted 20 times and incubated at 37 °C for 30 min. Digested tissues were cooled on ice before adding 2 mL cold 7.5 M ammonium acetate (Sigma) to precipitate proteins. Samples were vortexed at high speed for 10 s and then centrifuged at 4,000 g at 4 °C for 15 min. After the spin, the supernatant was transferred to a new 15 mL Falcon tube. 6 mL 100% isopropanol was added to each sample, which were then inverted 50 times and centrifuged at 4,000 g at 4 °C for 10 min. Genomic DNA pellets were visible as a small white pallet in each tube. The supernatant was discarded and 6 mL of 70% ethanol was added to wash the pellet. Each sample was inverted 10 times, and then centrifuged at 4,000 g at 4 °C for 5 min. The supernatant was discarded by pouring, and the remaining ethanol was removed using a pipette to completely remove any liquid. The precipitated genomic DNA was air dried for 30-60 min, and then resuspended in 0.5-1 mL nuclease-free water. Samples were then left overnight at room temperature. The next day, the gDNA solution was vortexed briefly and transferred to Eppendorf tubes. The gDNA concentration was measured using a Nanodrop (Thermo Scientific). For pre-injection cells, gDNA samples were extracted using Blood and Tissue kit (Qiagen) with multiple columns and pooled together for each individual sample. The sgRNA library readout was performed using a two-step PCR strategy, where the first PCR includes enough genomic DNA to preserve full library complexity (5 μg per reaction * 20 reactions = 100 ug gDNA per sample, 100 ug gDNA / 6.6 pg gDNA/cell / 70,290 sgRNAs > 200-fold coverage), and the second PCR adds appropriate sequencing adapters to the products from the first round PCR (2 μL per reaction, 6 reactions per sample). For PCR#1, a region containing sgRNA cassette was amplified using primers specific to the CRISPR library vector: Forward: 5'-gtgcgccaattctgcagacaaatgg-3' Reverse: 5'-caggatccaaaaaaagcaccgactc-3' PCR was performed using Phusion Flash High Fidelity Master Mix (ThermoFisher). For PCR#1, the thermocycling parameters were: 98 oC for 2 min, 25 cycles of (98 oC for 1 s, 62 oC for 5 s, 72 oC for 30 s), and 72 oC for 2 minutes. The final PCR products for each biological sample were pooled and used for amplification with barcoded second PCR primers (Supplementary Tables). Second PCR products were pooled and gel purified from a 2% E-gel EX (Life Technologies) using the QiaQuick Gel Extraction kit (Qiagen). The purified library was then quantified with a gel- based method using the Low-Range Quantitative Ladder (Life Technologies) and dsDNA High Sensitivity Qubit (Life Technologies). Libraries were sequenced with PhiX using an Illumina NovaSeq sequencer at the Yale Center for Genomic Analysis (YCGA).
identifiers.namespace: GEO
identifiers.id: GSM9282396_r1