title: GSM9663166: Day77_KO_R1; Homo sapiens; OTHER
library_construction_protocol: DNA extraction using the DNeasy Blood & Tissue Kit (#69504, Qiagen) Extracted DNA was subjected to PCR amplicon sequencing with unique molecular identifiers (UMIs) using a three-step PCR protocol. In the first step (PCR-1), UMI-containing primers (5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTACGTGACGTAGAAAGTAATAATTTCTTGGGT-3') and (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTN(25252525)NNNNNNNNNACTCGGTGCCACTTTTTCAA-3') were used for 4 cycles. PCR-2 utilized adaptor primers (5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') and (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3') for 22 cycles. PCR-3, performed by the sequencing facility, added sample-specific indexing in 7 additional cycles. The prepared libraries were sequenced on a NovaSeq platform with paired-end 150 bp reads, generating 10 million reads per sample at the Yale Center for Genomic Analysis (YCGA)