GSM8125104: Library2_plasmid; synthetic construct; OTHER
Genomic DNA was isolated using the NucleoSpin Blood XL Maxi kit (Machery-Nagel 740950.50) crRNA inserts were amplified from genomic DNA isolated from screens using 16 cycles of first round PCR using pooled 0-8nt staggered forward and reverse primers, treated with ExoSAP-IT (Thermo Fisher 78201.1.ML), followed by second round of PCR to introduce Illumina unique dual indices and adaptors. Sequencing primer binding sites, unique dual indices, P5 and P7 adaptor sequences are from Illumina Adaptor Sequences Document # 1000000002694 v16. PCR amplicons were subject to size selection by magnetic beads (SPRIselect, Beckman B23318) prior to sequencing on an Illumina NovaSeq6000 using SP100 kit (PE100) for Library 1 or SP500 kit (PE250) for Library 2. Sequencing of plasmid libraries were performed similarly, except 7 cycles of amplification were each used for Round 1 and Round 2 PCR. The size distribution of the final library was measured on an Agilent TapeStation system. We noted that even after magnetic bead selection of Round 2 PCR-amplified Library 2 plasmid library (colonies from which were Sanger sequencing verified) and genomic DNA from screens, smaller sized fragments from non-specific PCR amplification during Illumina sequencing library preparation persisted. This might contribute to the fraction of reads that could not be mapped to our reference 6-plex array. Thus, these unmapped reads do not necessarily reflect recombination of the crRNA library constructs, though the latter could contribute as well.