GSM5766304: Pre-kill_Daudi-Cas9; synthetic construct; OTHER
Cell pellets were lysed overnight at 66°C in 400 µL of cell lysis buffer (1% SDS, 50 mM Tris, pH 8, 10 mM EDTA) and 16 µL of sodium chloride (5 M), with 2.5 million cells per 416-µL lysis reaction. 8 µL of RNase A (10 mg/mL, Qiagen) was added to the cell lysis solution and incubated at 37°C for 1 hour. 8 µL of Proteinase K (20 mg/mL, Ambion) was then added and incubated at 55°C for 1 hour. 5PRIME Phase Lock Gel – Light tubes (Quantabio) were prepared by spinning the gel at 17,000xg for 1 minute. Equal volumes of the cell lysis solution and Phenol:Chloroform:Isoamyl alcohol (25:24:1, saturated with 10 mM Tris, pH 8.0, 1 mM EDTA, Sigma) were added to a 5PRIME Phase Lock Gel – Light tube. The tubes were vigorously inverted and centrifuged (17,000xg, 5 min, room temperature). The aqueous layer containing the genomic DNA above the gel was poured into DNA LoBind tubes (Eppendorf). 40 µL of sodium acetate (3 M), 1 µL of GenElute-LPA (Sigma-Aldrich), and 600 µL of isopropanol were added, and the solution was vortexed and frozen at -80°C. Once thawed, the solution was centrifuged at 17,000xg for 30 minutes at 4°C. After discarding the supernatant, the DNA pellet was washed with fresh room temperature ethanol (70%) and mixed by inverting the tube. The solution was then centrifuged at 17,000xg for 5 minutes at 4°C. The supernatant was removed and the DNA pellet was left to air dry for 15 minutes. The DNA Elution Buffer (Zymo Research) was added to the DNA pellet and incubated for 15 minutes at 65°C to resuspend the genomic DNA. A two-step PCR method was used to amplify and index the genomic DNA samples for Next Generation Sequencing (NGS). For the first PCR reaction, 10 µg of genomic DNA was used per 100-µL reaction (0.75 µL of Ex Taq polymerase, 10 µL of 10x ExTaq buffer, 8 µL of dNTPs, 0.5 µL of primer #1 (100uM), and 0.5 µL of primer #2 (100uM)) to amplify the integrated gRNA. The PCR #1 program was 5 min at 95°C; 28 cycles of 30 sec at 95°C, 30 sec at 53°C, 20 sec at 72°C; 10 min at 72°C. The PCR product solution was treated with SPRI purification (1.0X), and the DNA was eluted in 100 µL of water. To index the samples, 2 µL of purified PCR product (1:20 dilution) was used in a 50-µL PCR reaction containing 25 µL of Q5 Ultra II 2X MasterMix (NEB), 1.25 µL of Nextera i5 indexing primer (10 µM), and 1.25 µL of Nextera i7 indexing primer (10uM). The PCR #2 program was 3 min at 98°C; 10 cycles of 10 sec at 98°C, 10 sec at 62°C, 25 sec at 72°C; 2 min at 72°C. The final PCR product was treated with SPRI purification (0.7X), including two washes in 80% ethanol. DNA was eluted in 15 µL of water. The concentration was determined using a Qubit fluorometer (Thermo Fisher), and the library size was confirmed by gel electrophoresis and Bioanalyzer (Agilent). All indexed samples were pooled in equimolar amounts and analyzed by NGS.