title: NGS for CRISPRa Lentivirus
design: DNA was extracted from both puromycin only and puromycin + geneticin selected cell cultures with a QIAamp DNA mini kit (Qiagen).  Calabrese sgRNA template sequences were amplified from genomic DNA in 10 parallel 50 L 20 cycle PCR reactions/ sample. Each PCR reaction contained 1g genomic DNA, AmpliTaq Gold 360 Master Mix, staggered forward primers, and reverse primers. After 10 cycles, samples were then bead-purified using Omega Mag-Bind beads (Omega Biotek) and eluted in 100L. 10 L of this was then taken over into a second PCR reaction in which oligos were used that annealed to the Read 1 and Read 2 sequences, and contained overhangs that included illumina indexes and P5/P7 sequences. 10 cycles of PCR were performed. Samples were then pooled and resolved on a 1.5% agarose gel, purified, and sequenced on an Illumina NextSeq 500/550 with 75 cycles in the Read 1 orientation.