GSM9838042: NFkB_2_screen_A549_rep1; Homo sapiens; OTHER
Genomic DNA (gDNA) was isolated by lysing cell pellets overnight at 55°C in a buffer containing 10 mM Tris-HCl (pH 8.0), 5 mM EDTA, 100 mM NaCl, 1% SDS, and 0.5 mg/ml proteinase K. The lysate was then treated with RNase A (Qiagen) for 2 hours at 37°C. Subsequently, gDNA was purified through two sequential extractions with phenol:chloroform:isoamyl alcohol followed by a final extraction with chloroform:isoamyl alcohol. For barcode (BC) recovery, the entire gDNA yield from sorted/selected samples and 40 µg of gDNA from input samples were used as templates. To generate NGS libraries,  40 individual PCR reactions were performed for each gDNA sample. The PCR adds Illumina adaptors and indexes for each sample. Following amplification, all PCR reactions for a given sample were pooled. The pooled amplicons were concentrated and purified using the Monarch PCR & DNA Cleanup Kit (NEB), using six columns for each input sample and one column for each sorted/selected sample. The final purified barcode amplicons were gel-extracted and submitted for next-generation sequencing.