GSM1423055: GAF-RNAi replicate 1 (GRO-seq); Drosophila melanogaster; OTHER
Ten million nuclei were allowed to run-on for 10 minutes at 30°C in a reaction containing ATP, CTP, GTP, and BrUTP. The RNA was isolated using Trizol and acid phenol extractions. The RNAs were base hydrolyzed to an average length of 100-150 nucleotides, and RNAs with incorporated BrU were purified with 2 immunoprecipitations successive using Anti-BrdUTP agarose (Santa Cruz Biotechnology cat# sc32323 AC lot # I2111). The RNA was treated with PNK and polyadenylated using E. coli polyA polymerase to add 50-100 As to their 3’ ends. The polyadenylated RNA was reverse transcribed used with the following bar coded oligonucleotides (INOO3: 5’-pTAGAGATCGTCGGACTGTAGAACTCT-iSp18-CAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN, INOO4: 5’-pTGATGATCGTCGGACTGTAGAACTCT-iSp18-CAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN). The first 4 bases have the bar-code and iSp18 indicates an 18 carbon spacer between two oligo linkers used for Illumina sequencing. The resulting DNA was gel extracted and circularized using Epicentre CircLigase, and PCR amplified for 12-15 cycles. The cDNA was then sequenced on the Illumina Genome Analyzer.