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ABSTRACT: Transcriptomic of Galeruca daleruca 1st and 2nd instar larvae

PROVIDER: PRJNA1017636 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR26131801.fastq.gz	Fastqsanger.gz
	SRR26131801_1.fastq.gz	Fastqsanger.gz
	SRR26131801_2.fastq.gz	Fastqsanger.gz
	SRR26131802_1.fastq.gz	Fastqsanger.gz
	SRR26131802_2.fastq.gz	Fastqsanger.gz

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Project description:In this study we aimed to obtain gene expression data during development of Drosophila melanogaster. Therefore 10 critical stages from egg to adult fly were chosen. Specifically the purpose of the project was to study gene expression in the under-replicated regions of polytene chromosomes that were discovered earlier (see GSE2551). Biological samples from ten developmental stages were collected: 0-1 hour embryos, 5-6 hours embryos, 11-12 hours embryos, 1st instar larvae 0-1 hour after hatching, 2nd instar larvae after molt, 3rd instar larvae after molt, 0-1 hour prepupae, 13-14 hours pupae, adult males and adult females. 0-1h embryos demonstrate maternally expressed genes. 5-6h embryos represent early zygotic expression. 11-12h embryos represent organogenesis of larva. 1st instar larvae are freshly hatched completely formed larvae. 2nd instar larvae were collected after first molt. 3rd instar larvae were collected after second molt. 0-1h prepupae represent early metamorphosis. 13-14h pupae represent organogenesis of adult fly. Adult males and females collected separately represent differences between sexes. Embryos were collected as follows: flies were allowed to lay eggs for 45 min on the agar/apple juice medium with some yeast. Embryos were either collected immediately and frozen at -80°C or allowed to develop at 25°C to the stage of interest and then collected and frozen. 1st instar larvae were collected right after hatching. 2nd and 3rd larvae were collected after corresponding molts, 24 hours and 48 hours after hatching. 0-1 hour prepupae were picked each hour from the tube walls and either frozen at -80°C immediately or allowed to develop at 25°C to for 13 hours to obtain 13-14 hours pupae. Adult males and females were collected twice a day, kept for 3 days at 25°C and then separated and frozen at -80°C. The females collected in this way were mated. Total RNA isolation from the frozen samples was performed with TRIzol reagent (Invitrogen, #155-96-011). RNAs from ten stages were isolated in parallel and diluted to the same concentration (4ug/ul). The reference sample was prepared by mixing of the same amount of total RNA from each stage. 25ug of sample and reference RNAs were labeled with either Cy-3 or Cy-5 dUTPs (Amersham, #PA53022 and #PA55022) using SuperScript Direct cDNA Labeling System (Invitrogen, # L1015-02) according to provided protocol. Labeled cDNAs were purified using QIAquick PCR purification kit (QIAGEN, #28104) and combined in one tube. 20ug of sonicated salmon sperm DNA were added. DNA mixture was dried in the stream of nitrogen and dissolved in 60ul of hybridization buffer (50% deionized formamide, 6xSSC, 0.5% SDS and 5xDenhardt’s). Microarray slides were prehybridized in 6xSSC, 0.5% SDS and 1% BSA at 42°C water bath for 1 hour, rinsed with deionized water and dried in bucket rotor centrifuge in 50ml Falcon tubes (5 min, 1000xg). Labeled cDNAs were denatured at 100°C heat block for 2 min, cooled at room temperature, pipetted on the surface of microarray and covered with 25x60 mm LifterSlip (Erie Scientific Company). Assembled microarray was hybridized for 12-14 hours in the VersArray hybridization chamber (Bio-Rad) at 42°C water bath. After hybridization arrays were placed to the beaker with 1xSSC and 0.1% SDS to remove the LifterSlips. Then three washes were performed in the same solution (10 min, 5 min and 5 min) After that microarrays were rinsed with 1xSSC (30 sec), 0.1xSSC (30 sec) and dried in centrifuge as described above. Dry microarrays were scanned at 10 um resolution on ScanArray Lite instrument (Perkin Elmer). The scans were analyzed using TIGR Spotfinder program (www.tm4.org). Data were lowess normalized and filtered using MIDAS software (www.tm4.org). The final datasets were compiled in Microsoft Excel.

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