GSM3593878: HCT116_BRD4_Rep3; Homo sapiens; RNA-Seq
The UAS STAP-seq screening vector was generated from pSTAP-seq_fly-ctrl (Addgene ID 86380) by cloning an array of 4 upstream activating sequences (UAS) (obtained from pSGE_91_4xUAS_dCP (Addgene ID 71169)) upstream of the library insertion site (position of the core promoter) between the XhoI and BglII restriction sites. The core promoter candidate library was cloned from a pool of 12,000 synthesized 200-mer oligonucleotides obtained from Twist Biosciences Inc. The CP candidates spanned 133bp total, centered on the candidates major (+1) TSS, flanked by the Illumina i5 (33bp) and i7 (34bp) 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 CP 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 15bp to each of the adapters, serving as homology arms for directional cloning of the CP 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 UAS-STAP-seq screening vector was performed using In-Fusion HD (Clontech; cat. no. 639650) recombination.
GEO Accession
GSM3593878