title: GSM9102509: MPRA D6 NRF1 rep3; Homo sapiens; OTHER
library_construction_protocol: total RNA was isolated using Qiagen's RNeasy Plus kit (Qiagen, #74136) and mRNA subsequently purified using Dynabeads mRNA DIRECT purification kit (ThermoFisher, 61011). Libraries were prepared according to PMID: 34650237. Reverse transcription was performed with 500 ng mRNA, 100 nM primer and SuperScript IV (ThermoFisher, 18090050) according to the manufacturer's protocol. Reactions were incubated with 1 µL Themolabile Exonuclease I (NEB, M0568L) at 10 min at 37°, followed by inactivation at 85° for 5 min and cDNA purification with AMPure XP beads (Beckman Coulter, A63880) at a 1:1.1 sample-to-bead ratio. PCR amplification was performed in 50 µL reactions containing 5 µL cDNA, SYBR green (ThermoFisher, S7563), and PrimeStar Max DNA Polymerase (Takara, R045B). Reactions stopped in the early exponential phase, pooled, concentrated, gel purified and sequenced.