title: GSM8170172: COV434-STARR-seq-Output-1; Homo sapiens; OTHER
library_construction_protocol: The custom STARR-seq plasmid Library was transfected into COV434 cells with Lonza Nucleofector™ X Kit (SF-CM-138 ) according to the manufacturer's protocol. At 6 hours post-transfection, the media was removed and cells were washed with, washed with PBS, and total RNAs harvested using the Qiagen RNeasy system STARR-seq : Each washed cell pellet harvested from a 500 cm2 plate was lysed in a 50 mL conical tube with 5 mL of a 1:10 mixture of β-mercaptoethanol and Qiagen Buffer RLT. The lysates were vortexed, snap frozen, and stored at -80°C. Lysates were thawed on ice and passed through a 20-guage needle 10x. Total RNAs were purified with the Qiagen RNeasy midi-columns performing the on-column DNase step. The amount of purified RNA was quantified using a Nanodrop instrument, and RNA quality was assessed using an Agilent TapeStation 2200 with RNA ScreenTape. After adding 1 ul RNase Block (Agilent) samples were stored at -80. Total RNAs harvested from H295R or COV434 cells transfected with the custome STARR-seq plasmid library were subjected to poly-A enrichment, DNase digestion, reporter-specific reverse transcription, and PCR enrichment. ATAC-seq: Omni-ATAC protocol (Corces et al., 2017) was used, with the inclusion of the DNase treatment for COV434 cells.