Sequencing of Lentivirus-TKOV3 infected THP-1 Monocyte-Derived Macrophages
DNA was dissolved in 400 L DNA rehydration solution, quantified using a Qubit, and stored at -20C. Because the sorted cell pellets were fixed, a different protocol was necessary. They were incubated with 120 L 0.5 M EDTA, 500 L nuclei lysis mix, and 20 L 20 g/L proteinase K. The samples were incubated overnight at 55C. The next day 180 L 1 PBS was added to the mix. The protocol above was followed from the addition of RNAse A step with volumes scaled down 8-fold to account for fewer cells. After isolating the DNA, the region of DNA containing sgRNA sequences was amplified using PCR. 25 L NEBNext Ultra II Q5 Master, 2.5 L, and 10 M LCV2 forward and reverse primers were added to 3.5 g genomic DNA. The entire genomic DNA sample from the fixed sorted cells was used in the PCR. The following cycle was used for PCR amplification: 98C for 30 seconds, 30 cycles of 98C for 10 seconds, 66C for 30 seconds, and 72C for 15 seconds, and 72C for 2 minutes. Next, sequencing adaptors were added to the amplified sgRNA.5 L sgRNA product, 25 L NEBNext Ultra II Q5 Master, 2.5 L, 10 M Illumina TruSeq adapter i5, and Illumina TruSeq adapter i7 primers were combined and ran in a thermocycler: 98C for 30 seconds, 10 cycles of 98C for 10 seconds, 55C for 30 seconds, 65C for 15 seconds, and 65C for 5 minutes. The ligated sgRNA was purified via gel electrophoresis followed by purification using a PureLink Quick Gel Extraction Kit.