GSM8239954: Functional_screen_bin1_r1_DNA; Homo sapiens; OTHER
Following FACS sorting of cells based on eGFP/mCherry expression, genomic DNA was extracted from pelleted cells using the NucleoSpin Blood kit from Macherey Nagel, chosen based on cell quantity. Libraries were constructed using Illumina-compatible primers designed for Addgene #213963 plasmid, incorporating staggered regions to ensure cluster diversity. PCR amplification was performed using NEBNext Ultra II Q5 Master Mix, followed by product purification with the Select-a-Size DNA Clean & Concentrator MagBead Kit (Zymo Research). Quality was verified using Agilent TapeStation or BioAnalyzer. The libraries were sequenced to achieve a depth of >20 million reads per sample, with each sample uniquely barcoded using a combination of i5 and i7 indexes. This setup ensures sufficient coverage and identification of each library component in the pooled sample.