GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1357049

Query DataSets for GSM1357049

Status

Public on Mar 27, 2014

Title

S2_STARRseq_input

Sample type

SRA

Source name

whole genome library (in STARR-seq vector)

Organism

Drosophila melanogaster

Characteristics

cell type: S2 cells
genotype: y; cn bw sp - sequenced strain (Adams et al. 2000)

Treatment protocol

For all experiments cells were treated with ecdysone. The final concentration of ecdysone was 41 μM (10 mg of 20-hydroxyecdysone (Sigma; cat. no. H5142) per 500 ml of growth medium). After 24 hrs incubation with the hormone, cells were harvested.

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA (source: embryos of the sequenced strain: y; cn bw sp) was isolated, sheared and size selected (~500bp). Following the instructions of NEBNext® DNA Library Prep Reagent Set for Illumina® (NEB; cat. no. E6000L), Illumina Multiplexing Adapters (Illumina Inc; cat. no. PE-400-1001) were ligated and homology arms for In-Fusion® recombination were added by PCR, followed by recombination into the STARR-seq vector (pGL3-Promotor backbone (Promega; cat. no. E1751) with the sequence between BglII and FseI replaced with the following sequence, containing a Drosophila Synthetic Core Promoter (DSCP) (1), an ORF (sgGFP, Qbiogene, Inc), a ccdB suicide gene flanked by homology arms (used for cloning the genomic enhancer candidates during library generation), and the pGL3’s SV40 late polyA-signal.). The In-Fusion® reactions were transformed (MegaX DH10B; Invitrogen), grown in liquid culture and plasmids were isolated.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

PCR amplified Plasmid library, used for S2 STARR-seq
plasmid DNA (containing genomic DNA fragments)

Data processing

Library strategy: STARR-Seq
Basecall where performed using Real-Time Analysis (RTA) version > 1.12.4.2 or CASAVA 1.9.1
STARR-Seq:
Reads were mapped to the dm3 genome using bowtie (bowtie -p 12 -f -X 2000 -v 3 -m 1 --best --strata --quiet INDEX -1 reads_1.fa -2 reads_2.fa) excluding chrU, chrUextra, and chrM. For all subsequently analysis we used only reads collapsed on chromosome, start, end, strand (fragments). Significantly enriched regions (peaks) were called with an in-house pipeline using read density profiles of cDNA and input and an hypergeometric test to assign a p-value to each peak. Enrichment values were corrected within a 95% confidence interval taking into account the number of independent fragments at a single peak summit position.
RNA-seq:
Reads were mapped to the dm3 genome and transcriptome using bowtie (bowtie -f -v 3 -m 1 --best --strata --quiet -S INDEX -) and bfast reconciling the outputs. We estimated RPKM values for each gene based on the number of reads falling over each exon merging exons from a all genes' isoforms into one meta-transcript. Reads counts were normalized by the total reads per library and the merged meta-gene exons lengths.
Genome_build: dm3
Supplementary_files_format_and_content: All processed data files are in plain text. For STARR-seq we report for each peak the chromosome, the summit position, the enrichment over input (log2-scale) at the summit, and the p-value. For the RNA-seq data we report 3 different gene identifiers and the RPKM value for each gene.

Submission date

Mar 25, 2014

Last update date

May 15, 2019

Contact name

Daria Shlyueva

E-mail(s)

daria.shlyueva@imp.ac.at

Organization name

IMP

Lab

Stark lab