GSM5574556: Oligo_library_input_dev_rep2; synthetic construct; OTHER
24h after electroporation, total RNA was extracted using the RNeasy Maxi kit (Qiagen; cat. no. 75162), followed by polyA+ RNA isolation using Invitrogen Dynabeads Oligo(dT)25 (scaling up the manufacturer   s protocol accordingly; cat. no. 61005) and DNase treatment with Ambion Turbo DNase (cat. no. AM2239) at a concentration of at most 200 ng/  l for 45 minutes (min) at 37C. The reactions were then subjected to cleanup with Beckman-Coulter AMPure XP beads (Product No: A63881), at a ratio of 1:1.8. STARR-seq transcripts were specifically reverse transcribed using a gene specific primer (CTCATCAATGTATCTTATCATGTCTG), followed by RNaseA digest. After second strand synthesis (primer: TTGGTAAAGCCACCATGGAAAAG*G) we introduced a 10 bp unique molecular identifier (UMI) at the 3' end of the STARR-seq transcript (primer:  CAAGCAGAAGACGGCATACGAGATNNNNNNNNNNGTGACTGGAGTTCAGACGTGT*G), followed by a PCR step across the spliced intron junction of the mature STARR-seq transcript (primers: AAGCCACCATGGAAAAG*G*C*C*A*T and CAAGCAGAAGACGGCATACG*A). Samples were amplified for Illumina sequencing with an Illumina i5 adapter and a custom i7 index primer (CAAGCAGAAGACGGCATACGAGA*T). All PCR steps were performed using the KAPA HiFi HotStart ReadyMix (Roche; Material Number 7958927001), and cleaned up with Beckman-Coulter AMPure XP beads (Product No: A63881) at an appropriate ratio. See Neumayr et al., Curr. Protoc. Mol. Biol. 2019 for more details.