title: GSM3330959: SNU16 Input; Homo sapiens; OTHER
library_construction_protocol: Generation of plasmid libraries was based on the method described by Vanhille et al (39). Briefly, SNU16 genomic DNA was extracted using the PerfectPure DNA Cultured Cell kit (5 Prime). The DNA was sheared by sonication (Covaris S2) and size-selected on 1% agarose gels (350–650 bp). Size-selected DNA fragments were ligated to adaptors (Adaptor 1: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'; adaptor 2: 5'-GATCGGAAGAGCACACGTCT-3') after end repair and A-tailing (NEBNext Ultra End Repair/dA- Tailing and Ultra Ligation Module, New England Biolabs) and subjected to capture (NimbleGen SeqCap EZ, Roche). Post-capture DNA was PCR amplified using Q5 High-Fidelity DNA polymerase (NEB) to add homology arms for subsequent cloning (forward primer: 5'-GTAATAATTCTAGAGTCGGGGCGGGAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'; reverse primer: 5'- TATCATGTCTGCTCGAAGCGGCATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'; PCR program: 98 °C for 30 s; 17 cycles of 98 °C for 10 s, 65 °C for 30 s, and 72 °C for 30 s). The vector backbone was modified from pGL4.23[luc2/minP] (Promega). Briefly, the minP promoter was replaced with the SCP1 promoter and a CmR-ccdB cassette was cloned between luc2 ORF and SV40 poly (A). The PCR product was cloned into the vector backbone (linearized by SphI and NdeI restriction enzymes) through Gibson Assembly (Gibson Assembly Master Mix, New England Biolabs) and transformed into MegaX DH10B™ T1R electrocompetent cells (ThermoFisher Scientific). A total of 8 Gibson Assembly and transformations were performed. All transformed E. coli cells were pooled and cultured in 4 L LB medium and harvested at OD600 1.0. The input plasmid library was extracted using Plasmid Mega Kit (Qiagen) and drop dialyzed before transfection. Sequencing libraries were prepared from the input plasmid libraries by amplifying inserts from the plasmid DNA. 10 PCR reactions were set up, using Q5 High-Fidelity DNA polymerase (New England Biolabs), each with 1 ng plasmid as template (PCR program: 98 °C for 30 s; 12 cycles of 98 °C for 10 s, 65 °C for 30 s, and 72 °C for 30 s). Reactions were pooled, cleaned up and submitted for sequencing. Total RNA was extracted from transfected cells using PerfectPure RNA Cultured Cell Kit (5 Prime), followed by mRNA isolation using Dynabeads® Oligo (dT) kit (ThermoFisher Scientific) and treatment with TURBO DNase (ThermoFisher Scientific). Then target-specific first strand cDNA synthesis was performed with SuperScript III (ThermoFisher Scientific) to specifically reverse transcribe mRNA from the transfected plasmid library (RT primers: 5'-CAGTGACTGGAGTTCAGACG-3', 5'-GCAGCTTATAATGGTTACAAATAAAGC-3', 5'-ACATTTGTAGAGGTTTTACTTGCT-3', 5'-TCAATGTATCTTATCATGTCTGCTCGAAG-3'). After reverse transcription, reactions were treated with RNase A + H. Finally, the cDNA was PCR amplified using 5 μL cDNA reaction as template in every 50 μL PCR reaction. Q5 High-Fidelity DNA polymerase (New England Biolabs) was used, and the following PCR program: 98 °C for 30s; 20 cycles of 98 °C for 10 secs, 65 °C for 30 secs and 72 °C for 30 secs. Illumina HiSeq platform compatible primers were used (forward: 5'- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'; reverse: 5'-CAAGCAGAAGACGGCATACGAGAT-index-GTGACTGGAGTTCAGACGTG-3'). For each replicate, we set up separate final PCR reactions using 160 different indexing primers. PCR products for every 10 indexes were pooled and cleaned up by AmPure XP beads (Beckman Coulter; beads/reaction ratio = 0.8) and submitted for Illumina sequencing. CapStarr-seq