title: 5TagSeqFly_male_eGFP_nocap_3
organism: Drosophila melanogaster
description: Protocols: Cells were seeded in Nunc T-25 flasks at a concentration of 2.5 million cells in serum free Gibco Sneider 2.5 million cells were treated with 40 µg GFP dsRNA (approx 500bp) and harvested after 4 days of treatment. RNA was extracted with ALL prep DNA/RNA Mini Kit QIAGEN according to the manufacturer instructions. 20 µg of DNA-free total RNA was desphosphorylated by treatment with Calf Intestinal Alkaline Phosphatase, phenol purified and incubated with T4 Polynucleotide Kinase. Sample was phenol purified and ligated overnight using T4 RNA ligase 1 (NEB) to a DNA/RNA oligo (TACACTCTTTCCCTACACGACGCTCTTCCGATrCrUrXrXrXrXrXrX). Ligated total RNA was poly(A) purified, and reverse transcribed using random hexamers. Second strand was generated by 1 cycle PCR using oligo Biotin-TATAGCGGCCGCAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT. Double stranded cDNA was bound to Streptavidin beads, washed, end repaired, dA added and ligated to barcoded P7 linkers as described for the 3'T-fill protocol (PMID: 23295673). The PCR product was size-selected using double Ampure XP size and libraries with an average size of 440bp were submitted for Illumina 50bp paired-end sequencing.