title: Population2-5-1A_Generation3
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. Genomic DNA from the evolved populations was prepared using a Qiagen DNeasy Tissue Kit protocol (from Alexey Veraksa), with around 100 frozen flies (about 400 ul packed flies) per population. 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.
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: SAMEA14413883
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:Population2-5-1A_Generation3
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 3
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: population 2-5-1A, pool of around 100 flies
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-11768:Population2-5-1A_Generation3
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