title: GSM5583719: CREs to Barcode association; Escherichia coli; OTHER
library_construction_protocol: Plasmid DNA was purified from approximatly 1.2 million colonies using QIAprep Spin Miniprep Kit (Qiagen; Cat. N: 27106) according to the manufacturer instructions. 5 ng of the plasmid pool was used to attach P5 and P7 sequencing adapters in two separated PCRs. Bother PCRs were performed using the NEBNext® Ultra™ II Q5® Master Mix (New England Biolabs; Cat. N: M0544) with RV_univ_MPRA (CGACGCTCTTCCGATCT) + FWD_CRS_Tn7 (0.5µM each; GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCAGGACCGGATCAACT; PCR conditions: (98°C 30s; {98°C 10s, 65°C 30s, 72°C 3min} x3; 72°C 3min; Hold at 4°C) and P5NEXTPT5 (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG*A*T*C*T) + indexed Ad2.X (0.1 µM each; CAAGCAGAAGACGGCATACGAGAT XXXXXXXX GTCTCGTGGGCTCGGAGATGT; PCR conditions: 98°C 30s; {98°C 10s, 65°C 90s} x10; 72°C 5min; Hold at 4°C), respectively.