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Sample GSM1295426

Query DataSets for GSM1295426

Status

Public on Dec 19, 2013

Title

haltere_disc_rna (rep1 and rep2)

Sample type

SRA

Source name

3rd instar larval imaginal disc

Organism

Drosophila melanogaster

Characteristics

strain: y;cn,bw,sp
developmental stage: 3rd instar larvae
tissue: haltere disc

Growth protocol

Bottles were flipped every 12 hours, and aged at 25C until the designated time.

Extracted molecule

total RNA

Extraction protocol

Staged samples were homogenized in trizol, and total RNA was extracted, DNaseI treated, and purified. mRNA was purified with oligo(dT) beads and fragmented, followed by first and second strand cDNA synthesis.
1-100ng DNA was prepared for sequencing on an Illumina GAII or Hi-Seq machine at the UNC High-Throughput Sequencing Facility.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer II

Data processing

Basecalls performed with CASAVA version 1.7
FAIRE-seq GAII reads were filtered with Tagdust, and aligned to the Drosophila reference genome (version dm3) using Bowtie (version 0.12.3) with up to four possible alignments (m), and up to two mismatches in the seed (n) permitted.
FAIRE-seq HiSeq reads were first filtered with Tagdust. The first 8 bases were trimmed with fastx_trimmer to remove the internal Index. The last 6 bases were trimmed with fastx_trimmer to make HiSeq reads equivalent in length to GAII reads. Trimmed reads were aligned to the Drosophila reference genome (version dm3) using Bowtie (version 0.12.3) with up to four possible alignments (m), and up to two mismatches in the seed (n) permitted.
FAIRE-seq signal files were generated by extending each read to a total of 110bp, and the number of reads overlapping each base in the genome was counted.
FAIRE data were normalized by subtracting Input signal from FAIRE at each base, and z-scores were generated using the mean and standard deviation for each chromosome arm.
FAIRE peaks were called using MACS2 with Input as control, a shift size of 125bp, and a q-value cutoff of 1.00e-2.
RNA-seq GAII reads were filtered with Tagdust, and aligned to the Drosophila reference genome (version dm3) using TopHat (version 1.1.4) and default parameters.
RNA-seq HiSeq reads were first filtered with Tagdust. The first 8 bases were trimmed with fastx_trimmer to remove the internal index. The last 6 bases were trimmed with fastx_trimmer to make HiSeq reads equivalent in length to GAII reads. Trimmed reads were aligned to the Drosophila reference genome (version dm3) using TopHat (version 1.1.4) and default parameters.
Genome_build: dm3
Supplementary_files_format_and_content: FAIRE processed data files are in wig format, and contain Input-subtracted FAIRE z-scores, smoothed with a 200bp sliding window average and 10bp step size.
Supplementary_files_format_and_content: RNA processed data files are matrices of gene names with their associated FPKM values calculated with Cuffdiff from the Tophat-aligned reads and using a RefSeq genome annotation file.

Submission date

Dec 19, 2013

Last update date

May 15, 2019

Contact name

Daniel J McKay

E-mail(s)

dmckay1@email.unc.edu

Organization name

The University of North Carolina at Chapel Hill

Department

Biology, Genetics