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Sample GSM3109247

Query DataSets for GSM3109247

Status

Public on Jul 11, 2018

Title

Leg.Dll.ChIP2

Sample type

SRA

Source name

Third instar leg imaginal discs

Organism

Drosophila melanogaster

Characteristics

genotype: yw
developmental stage: L3
tissue: leg imaginal disc
antibody: ChIP with anti-Dll

Growth protocol

Wandering 3rd instar larvae were manually removed from Drosophila culture. Larvae were inverted in PBS to expose imaginal discs and fixed. Imaginal discs dissected off after fixation.

Extracted molecule

genomic DNA

Extraction protocol

Third instar larvae were dissected in PBS and fixed for 20 minutes at room temperature in 1.8% formaldehyde, 50mM HEPES, 1mM EDTA, 0.5mM EGTA, 100mM NaCl. After quenching (0.125M Glycine, 1xPBS, 0.01% Triton) the fixed tissue was washed first in Buffer A (10mM HEPES, 10mM EDTA, 0.5mM EGTA, 0.25% Triton, 1mM PMSF), then in Buffer B (10mM HEPES, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.01% Triton, 1mM PMSF), for 10 minutes at 4C. Imaginal discs were then dissected off the cuticles in Buffer B. Discs were sonicated in Buffer C (10mM HEPES, 1mM EDTA, 0.5mM EGTA, 1mM PMSF, plus complete protease inhibitor cocktail (Roche)) on ice/EtOH. Soluble chromatin was transferred to new tubes after centrifugation. Fresh chromatin, precleared with protein G sepharose beads (Roche), was incubated overnight at 4C with antibody in RIPA buffer (140mM NaCl, 10mM HEPES, 1mM EDTA, 1% Glycerol, 1% Triton X-100, 0.1% DOC, 1mM PMSF). Antibody-chromatin complexes were pulled down with protein G sepharose beads for 3 hours at 4C. Bound beads were washed 4-times in RIPA buffer (as above plus 0.1mg/mL ssDNA) and once in TE. Chromatin was eluted twice in Elution Buffer (1% SDS, 0.1M NaHCO3). 20ug Proteinase K was added, and samples were incubated for 3 hours at 55C, then 65C overnight. DNA was purified via phenol-chloroform extraction and ethanol precipitation.
DNA samples were prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Preparation Kit.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

ChIP with antibody against Drosophila Dll transcription factor
Leg.Dll.p2q4.peaks.txt

Data processing

ChIP-seq reads were aligned to the dm3 genome assembly using Burrows-Wheeler Aligner and replicates were merged.
Peaks were called, with input as control file, using MACS version 2.0.9 with the following setting: (P-value: 10e-2, mfold:2,50).
Peaks passing a more stringent q-value cutoff of 1.00e-04 were used for further analysis.
Genome_build: dm3
Supplementary_files_format_and_content: peak (bed format)

Submission date

Apr 24, 2018

Last update date

Jul 11, 2018

Contact name

Matthew Slattery

Organization name

University of Minnesota Medical School

Street address

1035 University Drive, SMed 219

City

Duluth