Sequencing of HEK-293 genomic TdT-catlayzed base addition sites
Sequencing methods were adapted from Kalhor et al 6 . Genomic DNA was recovered from frozen cell pellets using the Qiagen DNeasy Blood &amp; Tissue kit according to the manufacturers protocol. Recovered genomic DNA was eluted in nuclease free water. The hgRNA locus was amplified using the appropriate primer pair asnoted in Table S1 using the Biorad iQ SYBR Green Supermix and approximately 4-8 ng of the genomic DNA. Reactions were amplified by an initial 3 min, 95C denaturing step 7 followed by cycles of 10 s at 95 C and 30 s at 60C. Reactions were monitored in real time and cycling was stopped at mid-exponential amplification, typically 25 cycles. The products of the first reaction were used as a template for a second amplification with NEBNext Dual Indexing Primers. Each sample received a unique index. Reactions were. amplified using the same cycling conditions until mid-exponential phase, typically 7 cycles. An equal volume of each sample was pooled into a QC pool. The lengthdistribution and concentration of the library was determined using an Agilent 4200 Tapestation. Pool concentrations were further characterized by Qubit and qPCR methods. Sequencing was performed on an Illumina MiniSeq Mid Output flow cell. Libraries were supplemented with 15-20% phiX control library to increase clustering diversity. After demultiplexing, read counts for each sample were used to re-pool samples for a final sequencing run with evenly balanced indexing across all samples. The balanced pool was sequenced using the methods as before. Library preparation and sequencing were performed at the Rush Universtiy Genomics and Microbiome Core Facility.