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Sample GSM1482662

Query DataSets for GSM1482662

Status

Public on Mar 26, 2015

Title

Df(3L)ED217_M_Df(3L)/+_rep_3 [Sample_310603]

Sample type

SRA

Source name

Head

Organism

Drosophila melanogaster

Characteristics

drosdel deficiency: Df(3L)ED217
developmental stage: Adult
Sex: M
x chromosome dose: 1
replicate: 3
genotype: Df(3L)ED217/+
flag: pass
crosses: F0 = w1118/w1118;+/+;Df(3L)ED217/TM3 X w1118/Y;+/+;+/+. F1 (sample) = w1118/Y;+/+;Df(3L)ED217/+

Growth protocol

Flies were grown at 25°C on cornmeal media Fly Food A (LabExpress, Ann Arbor, MI). Adults were aged 5 days before dissection.

Extracted molecule

total RNA

Extraction protocol

We used RNeasy 96 Kit (Qiagen, Valencia, CA) to extract total RNA from a pool of 10 dissected heads for each sample. Frozen heads were added to 2ml Axygen 96-well plates (Corning, Life Sciences, Union City, CA) pre-loaded with 200 µL 1mm glass beads (Biospec Products, Inc., Bartlesville, OK) and 200 µL RLT Buffer (Qiagen, Valencia, CA). After covering the plates with Axygen sealing mats, we homogenized the samples with Mini-BeadBeater-96 (Biospec Products Inc., Bartlesville, OK). Manual purification was carried out using QIAvac 96 vacuum manifold (Qiagen, Valencia, CA).
We followed TruSeq RNA Sample Preparation V2 high-throughput protocol (Illumina Inc., San Diego, CA) to construct RNA-seq libraries from polyA+ RNA. For each sample, 100ng of total RNA was used as input in the initial step and 10pg ERCC spike-in RNA (Zook et al. Plos ONE 2012, 7(7): e41356., pool 78A) was added to the Elute, Prime, Fragment Mix in the "Purify and fragment mRNA” step.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Processed data files:
cufflinks_fpkm_gene_ERCC_249.txt
DESeq2_normalized_read_counts_gene_249.txt
HTSeq_raw_read_counts_gene_ERCC_249.txt
cufflinks_fpkm_gene_235.txt
DESeq2_normalized_read_counts_gene_235.txt
HTSeq_raw_read_counts_gene_ERCC_235.txt

Data processing

Basecalls were performed using CASAVA-1.8.2/RTA 1.17.21.3/HiSeq Control Software 2.0.10.0 on an Illumina HiSeq 2000 (Illumina Inc., San Diego, CA).
We mapped RNA reads to D. melanogaster genome (release 5, from the FlyBase, excluding “U” or “Uextra”), along with the 96 ERCC spike-in sequences (Zook et al. Plos ONE 2012, 7(7): e41356), with Tophat (v2.0.10) (Kim et al. Genome Biology 2013, 14:R36). We used parameter -g 1 to retain only uniquely mapped reads and provided a GTF format annotation with the -G option. The GTF format annotation includes the annotation for genes on existing chromosomes (derived from FlyBase annotation release 5.57 in GFF format, excluding genes on “U” or “Uextra”), as well as the annotation for 96 exogenous controls.
We measured gene expression levels using cufflinks v2.1.1 (Trapnell et al. Nature Protocols 2012, 7(3):562-78) in Fragments Per Kilobase of transcript per Million mapped reads (FPKM). We also measured the gene expression level in normalized read counts using HT-Seq v0.5.4p1 (Anders et al. Bioinformatics 2015, 31(2):166-9) and R package DESeq2 v1.6.3 (Love et al. Genome Biology 2014, 15(12):550).
There are two sets of processed data. One includes all 249 samples. The other was filtered in two steps (see "flag” for each sample) to make sure every DrosDel deficiency (Df) had at least two reliable replicates in each karyotype/sex combination. First, we checked to see if genes within the reported DrosDel deficiencies (Dfs) had reduced expression, and removed samples without expression reduction at the Dfs; 2) We removed samples without replicates in any one of the three karyotype/sex combinations (XX female, XX female transformed to male and XY male). The remaining 235 samples include 113 samples with 19 Dfs on the X chromosome, 99 samples with 11 Dfs on the autosome 3L, and 23 controls without Dfs.
Genome_build: Drosophila melanogaster Release 5
Supplementary_files_format_and_content: DESeq2_normalized_read_counts_gene_235.txt contains a matrix with read counts of all the genes normalized by DESeq2 for the 235 samples that have replicates and validated deletions. Column names in the file correspond to sample names, row names correspond to FlyBase gene_ID.
Supplementary_files_format_and_content: DESeq2_normalized_read_counts_gene_249.txt contains a matrix with read counts of all the genes normalized by DESeq2 for all the 249 samples. Column names in the file correspond to sample names, row names correspond to FlyBase gene_ID.
Supplementary_files_format_and_content: HTSeq_raw_read_counts_gene_ERCC_235.txt contains matrix with raw read counts of all the genes and spike-ins for the 235 samples that have replicates and validated deletions. Column names in the file correspond to sample names, row names correspond to FlyBase gene_ID and ERCC ID.
Supplementary_files_format_and_content: HTSeq_raw_read_counts_gene_ERCC_249.txt contains matrix with raw read counts of all the genes and spike-ins for all the 249 samples. Column names in the file correspond to sample names, row names correspond to FlyBase gene_ID and ERCC ID.
Supplementary_files_format_and_content: cufflinks_fpkm_gene_235.txt contains matrix with FPKM values of all the genes in the 235 samples that have replicates and validated deletions. Column names in the file correspond to sample names, row names correspond to FlyBase gene_ID.
Supplementary_files_format_and_content: cufflinks_fpkm_gene_ERCC_249.txt contains matrix with FPKM values of all the genes and spike-ins in all the 249 samples. Column names in the file correspond to sample names, row names correspond to FlyBase gene_ID and ERCC ID.

Submission date

Aug 20, 2014

Last update date

May 15, 2019

Contact name

Brian Oliver

E-mail(s)

briano@nih.gov

Phone

301-204-9463

Organization name

NIDDK, NIH