title: GSM4038039: DNA_vector v1; synthetic construct; RNA-Seq
library_construction_protocol: Lysates were DNA depleted using a gDNA eliminator column (Qiagen) and RNA was extracted using a RNeasy Plus micro kit according to the manufacturer's instructions (Qiagen). For library preparation, 1μg RNA was treated with TURBO DNAse (ThermoFisher) and reverse transcribed (SuperScript IV VILO, ThermoFisher) according to the manufacturer's instructions. DNA removal was confirmed by qPCR for GFP and compared to a no-RT control (all performed in triplicate). Sequencing libraries were prepared by PCR amplification (30 cycles, annealing temperature 55C) using PfuUltra II Fusion DNA polymerase (Agilent) and custom primers that were designed to anneal to a 3' site within the EGFP gene (F) and the 3' universal primer site within the oligonucleotide sequences (R). These primers contained sequencing indices to enable multiplexing. Amplified libraries were cleaned using sequential SPRI bead clean-up (0.6X, 1.6X, 1.0X; Agencourt AMPure XP, Beckman Coulter). 4 sequencing libraries were made from the MPRA vector using 50ng input and 18 cycles of PCR (other conditions were the same as for the RNA libraries).
attributes.tag: GEO Accession
attributes.value: GSM4038039