GSM9105688: RSL3-1; Homo sapiens; OTHER
Genomic DNA was extracted from over 3×10⁷ cells per sample using standard phenol-chloroform extraction, followed by ethanol precipitation. DNA quality was assessed using agarose gel electrophoresis, and concentration was quantified using a Qubit fluorometer. At least 3 μg of genomic DNA per sample was used for sgRNA library amplification. Two-step PCR was performed to amplify sgRNA cassettes. The first PCR used NEB Q5 polymerase to amplify sgRNA regions from genomic DNA (10,000× coverage), followed by magnetic bead purification. A second PCR added Illumina index primers. Libraries were size-selected (~280 bp), quantified (Qubit), and adjusted to 3 ng/μL. Final libraries were pooled and sequenced on Illumina HiSeq Xten or NovaSeq platforms (PE150, paired-end 150 bp).