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Sample GSM1513726

Query DataSets for GSM1513726

Status

Public on Dec 10, 2014

Title

X16_BAC_STARRseq_input

Sample type

SRA

Source name

focused BAC library (in STARR-seq vector)

Organism

Drosophila melanogaster

Characteristics

genotype: y; cn bw sp - sequenced strain (Adams et al. 2000)
experimental procedure: STARR-Seq

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. To construct the dCP STARR-seq vector, the sequence between BglII and FseI from the pGL3-Promoter backbone (Promega; cat. no. E1751) was replaced with the Drosophila Synthetic Core Promoter (DSCP), 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 hkCP STARR-seq vector is identical to the dCP STARR-seq vector except we replaced the DSCP with the RpS12 core promoter. 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

plasmid DNA (containing genomic DNA fragments)
PCR amplified Plasmid library

Data processing

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 4 -f -X 2000 -v 3 -m 1 --best --strata --quiet INDEX -1 reads_1.fa -2 reads_2.fa) excluding chrU, chrUextra, and chrM. We additionally employed redundancy filter to remove clustered reads that could be due to sequence artefacts. For all subsequently analysis we used only reads collapsed on chromosome, start, end, strand (fragments), and merged these fragments from the two replicates. Significantly enriched regions (peaks) were called with an in-house pipeline using read density profiles of STARR-seq and input. We performed hypergeometric tests 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.
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 at the summit, and the p-value.

Submission date

Sep 25, 2014

Last update date

May 15, 2019

Contact name

Alexander Stark

Organization name

Research Institute for Molecular Pathology (IMP), Vienna Biocenter VBC

Street address

Dr Bohr-Gasse 7

City

Vienna