title: Fly2-1d_Generation1
organism: Drosophila melanogaster
description: Protocols: Flies were maintained under standard lab conditions (25°C). Adult flies were collected and frozen at -20°C prior to DNA extraction. Each fly was squished and incubated at 37 °C for 30 min in Squish Buffer (10 mM Tris pH 8.0, 1 mM EDTA, 25 mM NaCl, 0.15 mg/ml Proteinase K), followed by a clean-up with a Genomic DNA Clean Concentrator kit (Zymo Research). 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, 0.286 ng and 0.5ng 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 instrument 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-05-16T00:17:20Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-05-16T00:17:20Z
attributes.tag: External Id
attributes.value: SAMEA14413847
attributes.tag: INSDC center name
attributes.value: EMBL
attributes.tag: INSDC first public
attributes.value: 2023-05-16T00:17:20Z
attributes.tag: INSDC last update
attributes.value: 2023-05-16T00:17:20Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11768:Fly2-1d_Generation1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: fruit fly
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: generation
attributes.value: generation 1
attributes.tag: genetic modification
attributes.value: ethyl methanesulfonate induced mutation at G0
attributes.tag: genotype
attributes.value: VK33 inserted bicoid-EGFP
attributes.tag: individual
attributes.value: fly d from pool 2-1
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-11768:Fly2-1d_Generation1
attributes.tag: scientific_name
attributes.value: Drosophila melanogaster
attributes.tag: sex
attributes.value: mixed
attributes.tag: specimen with known storage state
attributes.value: frozen specimen
attributes.tag: strain
attributes.value: VK33