GSM7050445: HARPE on DPR - replicate 2 in Drosophila (in vitro); Drosophila melanogaster; OTHER
The HARPE procedure and data processing was performed for the DPR as described in Vo ngoc et. al (2020), with the exception that the in vitro transcription reactions were carried out with Drosophila nuclear extracts by the method of Wampler et al. (1990). A brief summary is as follows. The HARPE plasmid library was subjected to in vitro transcription (six standard reactions, 300 ng DNA each) with Drosophila extracts for 30 minutes. The transcription was performed independently two times to ensure reproducibility of the data. The combined RNA transcripts from the six reactions were extracted with Trizol™ LS (Thermo Fisher Scientific) and the contaminating plasmid DNA was removed with the TURBO DNA-free™ Kit (Thermo Fisher Scientific). After ethanol precipitation, the RNA was subjected to reverse transcription with SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific) and treatment with RNase H (New England Biolabs). The cDNA was extracted with phenol-chloroform-isoamyl alcohol, precipitated with ethanol, and size-selected on a 6% polyacrylamide-8M urea gel with radiolabeled size markers. The HARPE plasmid DNAs and the size-selected cDNAs were used as templates to generate DNA amplicons for Illumina sequencing by using custom forward oligonucleotides containing the Illumina P5 and Read1-primer sequences preceding the sequence corresponding to nucleotides +1 to +16 (relative to the +1 TSS) of the SCP1m promoter cassette. Reverse primers were selected from the NEBNext® Multiplex Oligos for Illumina® kits (New England Biolabs), which match the Illumina Read2-primer sequence present on the HARPE plasmid and corresponding cDNA. NGS PCR amplicons were size-selected on native 6% polyacrylamide gels prior to Illumina sequencing. As described above, the HARPE plasmid DNAs and the size-selected cDNAs were used as templates to generate DNA amplicons for Illumina sequencing by using custom forward oligonucleotides containing the Illumina P5 and Read1-primer sequences preceding the sequence corresponding to nucleotides +1 to +16 (relative to the +1 TSS) of the SCP1m promoter cassette. Reverse primers were selected from the NEBNext® Multiplex Oligos for Illumina® kits (NEB), which match the Illumina Read2-primer sequence present on the HARPE plasmid and corresponding cDNA. NGS PCR amplicons were size-selected on native 6% polyacrylamide gels prior to Illumina sequencing.