title: GSM8371613: WHR-OUTPUT-rep02-clean; Homo sapiens; OTHER
library_construction_protocol: Total RNA was extracted using Monarch Total RNA Miniprep Kit (New England Biolabs), and treated with TURBO DNA-free Kit (Thermo Fisher) following the manufacturer's protocol. We then isolated the poly(A) RNA using NEB Next Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) and precipitated each replicate using Monarch RNA Cleanup Kit (New England Biolabs). The plasmid library was used to construct input sequencing library before cell transfection. In brief, 100ng plasmid library was treated with RT-UMI as following: 98°C for 30s, 63°C for 30s and 72°C for 2 min. Subsequently, two rounds of amplification were applied using Vector-F and Read1-R or barcode primers, respectively. On the other hand, total RNA was extracted and treated with DNase I for output sequencing construction after 24 h post-transfection. The poly(A) RNA was further isolated for cDNA synthesis (NEB, USA). Next, specific reverse transcription was performed using RT-UMI  (NEB, USA), and treated with RNase A (Takara, Japan). Similarly, two rounds of amplification were performed using junction primer  and Read1-R or barcode primers, respectively.