GSM8174096: B16F10_m11_Spleen; Mus musculus; OTHER
Genomic DNA was extracted from spleens, dissected tumors, and pre-injected cell pellets using the methods from a previously study 33. Briefly, each sample was put in a 15 mL Falcon tube and had 6 mL Lysis Buffer (50 mM Tris, 50 mM EDTA, 1% SDS, pH adjusted to 8.0) and 30 μL of 20 mg/mL Proteinase K (Qiagen) added, then incubated at 55 °C overnight. After tissue digestion, 30 μL of 10 mg/mL RNase A (Qiagen) was added to the lysed sample 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 mixed thoroughly and then centrifuged at 4,000 x g at 4 °C for 15 min. The supernatant was moved to a new 15 mL Falcon tube, 6 mL 100% isopropanol was added, and samples were centrifuged at 4,000 x g at 4 °C for 10 min. Genomic DNA pellets were washed once with 70% ethanol, and then centrifuged at 4,000 x g at 4 °C for 5 min. The supernatant was discarded, and remaining ethanol was removed using a pipette. Genomic DNA was air dried for 30-60 min, and then resuspended in 0.5-1 mL nuclease-free water overnight at room temperature. For cell pellets, 100-200 μL QuickExtract solution (Epicentre) was directly added to cells and incubated at 65 °C for 30 min. For mouse lymph nodes, QIAmp Fast DNA Tissue Kit (Qiagen) was used for gDNA extraction following the manufacturer's protocol. Library readout was performed by nested PCR reactions to decrease the effect of PCR-amplification bias on the screen. The first-round PCR amplified the sgRNA sequence of the AAV-SB vector from genomic DNA (~2.5 μg per 50 μL reaction, 24 reactions per sample), and the second-round PCR added barcoded sequencing adapters (2 μL per 30 μL reaction, 6 reactions per sample). Primers for PCR#133: Forward: 5'-aatggactatcatatgcttaccgtaacttgaaagtatttcg-3' Reverse: 5'- actcctttcaagacctagtcgacg-3' Each round of PCR was performed using the following thermocycler conditions: 98 °C for 1 min, 25 cycles of (98 °C for 1s, 60 °C for 5 s, 72 °C for 12 s), and 72 °C for 2 min. All PCR reactions were performed using Phusion Flash High Fidelity Master Mix. The final PCR products were pooled and normalized for each biological sample before combining individual biological samples. The pooled product (150 ng per sample) was then gel purified from a 2% E-gel EX (Life Technologies) using the QiaQuick Gel Extraction kit (Qiagen). The purified, pooled library was then quantified with a gel-based method using the Low-Range Quantitative Ladder (Life Technologies), dsDNA High-Sensitivity Qubit (Life Technologies), BioAnalyzer (Agilent), and/or qPCR. Libraries were sequenced with 5-20% PhiX using an Illumina NovaSeq 4000 sequencer at the Yale Center for Genomic Analysis (YCGA).