GSM6482115: ced-6 enhancer libraries rep2 input; 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. Random 8nt variant libraries were generated using a PCR approach with degenerate oligonucleotides. Forward primers were designed to anneal directly downstream of the enhancer position of interested followed by 8 degenerate bp (creating 65,536 variants) and another 20 bp complementary stretch. Reverse primers were complementary to the 20 bp 5' of the degenerate stretch. The STARR-seq vector containing the wildtype enhancer of interest (either ced-6 or ZnT63C) was used as a template for the PCR. The PCR was run across the whole STARR-seq plasmid, followed by DpnI digest and a Gibson reaction that re-circularizes the plasmid. Libraries were grown in 2l LB-Amp (final ampicillin concentration 100µg/mL). Variant libraries of the same enhancer i.e. ced-6 enhancer pos110, pos182, pos230, pos241 and ZnT63C enhancer pos142, pos180, pos210 were pooled to equimolar ratio, together with another synthetic oligo library containing wt enhancer sequences and negative regions. STARR-seq