GSM4743373: Med14_msAuxin_rep3 [STARR-seq]; Homo sapiens; OTHER
After 6h of treatment cells were harvested and total RNA was isolated using RNeasy Maxi kit (Qiagen #75162) containing beta-Mercaptoethanol supplemented RLT buffer. Spike-in control was added in a 1:1000 ratio to the isolated total RNA. polyA+ mRNA was isolated via Oligo-dT25 beads  (Invitrogen #61005) followed by 1h 37°C TurboDNase I treatment (Invitrogen #AM2238). Isolated mRNA was cleaned via AMPure XP beads (Beckman Coulter #A63882) 1:1.8 ratio (RNA:beads) followed by reverse transcription utilizing SuperScript III (Invitrogen #18080093) using a gene specific primer (GSP): 50°C for 1h, 70°C for 15min, 4°C for 10min. cDNA was treated with RNaseA (Thermo Fisher #EN0531), 37°C for 1h followed by cleanup via AMPure XP beads 1:1.8 ratio. Next, junction PCR which allows enrichment of reporter transcripts was performed using KAPA 2x HiFi (KapaBiosystems #KK2601) and “junction primers” spanning over the spliced intron utilizing the thermocycler program: 98°C - 45 sec, 98°C - 15 sec, 65°C - 30 sec 16 cycles, 72°C - 70 sec, 72°C - 120 sec followed by purification with AMPure XP 1:0.8 ratio (DNA:beads). Sequencing-ready PCR was performed on the junction PCR products using Illumina primers with the thermocycler program: 98°C - 45 sec, 98°C - 15 sec, 65°C - 30 sec 5 cycles, 72°C - 45 sec, 72°C - 120 sec. Illumina adapter containing STARR-seq library fragments were cleaned using SPRIselect beads (Beckman Coulter #B23318) with a stringent ratio of 1:0.5 (DNA:beads) and subjected to paired end deep sequencing.