title: GSM5582239: STAPseq_BRD4_msAuxin_rep2; Homo sapiens; OTHER
library_construction_protocol: The TATA-box + CCAAT-box promoter candidate library was cloned from a pool of 2,000 synthesized 300-mer oligonucleotides (Twist Biosciences Inc) into a human STAP-seq screening vector (Addgene #). The candidate sequences spanned 240bp in total, with 205bp upstream and 35bp downstream of the selected TSSs, followed by a 10bp random barcode, and flanked by the Illumina i5 (25bp) and i7 (25bp) adapter sequences upstream and downstream, respectively, serving as constant linkers for amplification and cloning. To comprehensively amplify the oligonucleotide pool (diluted to 1ng/µl) and thereby create the candidate library insert we performed 20 PCR reactions (98°C for 45 seconds (s); followed by 15 cycles of 98°C for 15s, 65°C for 30s, 72°C for 10s) with 1µl diluted oligonucleotide pool as template, using KAPA Hifi Hot Start Ready Mix (KAPA Biosystems; cat. no. KK2602) and primers (fw: TAGAGCATGCACCGGACACTCTTTCCCTACACGACGCTCTTCCGATCT and rev: GGCCGAATTCGTCGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) that add extra 23bp to each of the adapters, serving as homology arms for directional cloning of the candidate library insert using In-Fusion HD (Clontech; cat. no. 639650). All PCR reactions were pooled and purified with Agencourt AMPureXP DNA beads (ratio beads/PCR 1.4; cat. no. A63881). Directional cloning of the library insert (amplified oligonucleotide pool) into the STAP-seq screening vector was performed using In-Fusion HD (Clontech; cat. no. 639650) recombination.