Whole genome sequencing of TF-AID-mutagenized flies
Whole genome sequencing of TF-AID-mutagenized flies
Flies were maintained under standard lab conditions (25°C). Adult flies were collected and frozen at -20°C prior to DNA extraction. Genomic DNA was prepared using a Qiagen DNeasy Tissue Kit protocol (from Alexey Veraksa). Sequencing libraries were prepared based on publications by Picelli et al., 2014 (Smartseq2, Nature Protocols, 9(1): 171) and Hennig et al. 2018 (G3, 8:79). In short, 1.25 ng of each sample was taken into a tagmentation reaction containing 1.25 uL of Dimethylformamide, 1.25 uL of tagmentation buffer (40 mM Tris-HCl pH 7.5, 40 mM MgCl2) and 1.25 uL of an in-house generated and purified Tn5 (previously prepared as described by Hennig et al. 2018 (G3, 8:79) and diluted to a 1:50 ratio with water). The mixture was incubated at 55 degrees for 3 minutes. After that, 1.25 uL of SDS 0.2% was added to stop the tagmentation reaction followed by a 5 minutes incubation at room temperature. The resulting fragments were then amplified by PCR using 6.75 uL of KAPA 2 X HiFi master mix, 0.75 uL of Dimethyl sulfoxide and 2.5 uL of dual indexed primers. Cycling conditions were as it follows: 3 minutes at 72°C; 30 seconds at 95°C; 12 cycles of 20 seconds at 98°C, 15 seconds at 58°C, 30 seconds at 72°C; 3 minutes at 72°C. The PCR products were then purified using a magnetic SPRI bead purification (0.8X). Libraries size and molarity were assessed using Bioanalyser and individual libraries were pooled. Finally, the pool was size selected using two rounds of magnetic SPRI bead purification (0.65X) and quantified using the Qbit dsDNA High Sensitivity assay.