GSM6482108: LibFR013_Twistmix2_input_dev_rep1; Drosophila melanogaster; 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/µl for 30 minutes (min) at 37°C. The reactions were then subjected to Qiagen RNeasy MinElute reaction clean-up (cat. no. 74204), for Turbo DNase inactivation and RNA concentration.  After reverse transcription and second strand synthesis a unique molecular identifier (UMI) was added to each transcript. This is followed by two nested PCR steps, each with primers that are specific to the reporter transcripts such that STARR-seq does not detect endogenous cellular RNAs. See Neumayr et al., Curr. Protoc. Mol. Biol. 2019. Oligo libraries were synthesized by Twist Bioscience including 249 bp enhancer sequence and adaptors for library cloning. Following the instructions of NEBNext® DNA Library Prep Reagent Set for Illumina® (NEB; cat. no. E6000L), Illumina Multiplexing Adapters (Illumina Inc; cat. no. PE-400-1001) were ligated and homology arms for In-Fusion® recombination were added by PCR, followed by recombination into the STARR-seq vector. To construct the dCP STARR-seq vector, the sequence between BglII and FseI from the pGL3-Promoter backbone (Promega; cat. no. E1751) was replaced with the Drosophila Synthetic Core Promoter (DSCP), an ORF (sgGFP, Qbiogene, Inc), a ccdB suicide gene flanked by homology arms (used for cloning the genomic enhancer candidates during library generation), and the pGL3's SV40 late polyA-signal. The hkCP STARR-seq vector is identical to the dCP STARR-seq vector except we replaced the DSCP with the RpS12 core promoter. The In-Fusion® reactions were transformed (MegaX DH10B; Invitrogen), grown in liquid culture and plasmids were isolated.  See Neumayr et al., Curr. Protoc. Mol. Biol. 2019. STARR-seq