GSM8014849: Reporter RNA-seq from synthetic insulator screen, rep1; Drosophila melanogaster; RNA-Seq
Total RNA was isolated from S2R+ cells 72h after transfection. Cells were lysed in TRIzol LS reagent (ThermoFisher 10296028) following the manufacturer's protocol. RNA was precipitated with isopropanol, resuspended in nuclease-free water, treated with DNase (ThermoFisher AM2238), purified using RNAClean XP beads (Beckman Coulter A63987), and eluted in nuclease-free water. PCR-amplicon libraries of eGFP and mCherry mRNA 3'UTR fragments comprising a unique shared barcode were generated from total RNA using a reverse transcriptase primer complementary to the SV40 3'UTR sequence of both eGFP and mCherry reporter genes (for the BX-C locus screen) or a reverse transcriptase primer with an "8N" unique molecular identifier (UMI) (for the eve locus and synthetic insulator screens) using SuperScript IV Reverse Transcriptase (ThermoFisher 18090010) according to the manufacturer's protocol. Sequencing-ready libraries were then generated by two successive PCR reactions using KAPA HiFi HotStart ReadyMix (KAPA Biosystems KK2602). DNA-seq libraries for all insulator-seq screens were also prepared by two successive PCRs starting from the original insulator reporter plasmid library as template.