title: TDS of NIH/3T3: Cas9 mRNA/sgRNA ratio screening
design: PCR amplicons underwent end repair, 5 phosphorylation, and dA-tailing in a single reaction, followed by T-A ligation to attach sequencing adaptors to both ends. A secondary PCR was carried out using P5 and P7 primers to introduce unique index sequences and flow cell-binding sites for multiplexed sequencing. Final libraries were purified with magnetic beads and quality-checked prior to sequencing. Paired-end sequencing (150 bp) was conducted on the NovaSeq 6000 platform (Illumina, CA, USA).