GSM7746161: LacZRNAi biological rep1; Drosophila melanogaster; RNA-Seq
RNA was purified using the Maxwell RSC simply RNA tissue extraction kit (Promega) with on-bead DNase I digestion. The RNA concentration was determined using a NanoDrop spectrophotometer. RNA samples were submitted for Agilent Tapestation analysis and RNA integrity number (RIN) values were 8.7-10. Total RNA was poly(A) selected using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolab). We reverse transcribed 1 ug of total RNA with the partial P7 adapter (Illumina_4N_21T) and dNTPs with the addition of spiked-in azido-nucleotides (AzVTPs) at 5:1. We click-ligated the p5 adapter (IDT) to the 5′ end of the cDNA with CuAAC. We then amplified the cDNA for 21 cycles with Universal primer and 3′ indexing primer and purified it on a 2% agarose gel by extracting amplicon from 200-300 base pairs. We pooled the libraries and sequenced single-end, 75 base-pair reads on a Nextseq 550 (Illumina).