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Sample GSM3426142

Query DataSets for GSM3426142

Status

Public on Apr 25, 2019

Title

young_yw_ChIP_rep2

Sample type

SRA

Source name

Whole Body

Organism

Drosophila melanogaster

Characteristics

tissue: Whole Body
developmental: 14 days after eclosion
condition: young
Sex: Female
genotype: yw;+;+

Treatment protocol

Larvae were reared on normal food. Eclosed flies were collected over the course of 2 days and allowed to mate before separation. 25-30 females were collected per food vial and aged under standard conditions. Flies were transferred to new food every 3-4 days until reaching the age for extraction.

Growth protocol

Flies were maintained at 25°C, 40% relative humidity and 12-hour light/dark. Adults were reared on agar-based diet with 0.8% cornmeal, 10% sugar, and 2.5% yeast

Extracted molecule

genomic DNA

Extraction protocol

~200 female flies were anesthetized with Flynap (Carolina, Burlington, NC) and ground into a powder in liquid nitrogen. Crosslinking was allowed for 20 minutes in 1X PBS with 1% paraformaldehyde before quenched with glycine. Chromatin was washed several times with 1X PBS supplemented with protease inhibitor. Pellets were washed once with cold cell lysis buffer (5mM HEPES pH7.6, 100mM NaCl, 1M EDTA,0.5% NP-40, ddH2O, 0.1% PIC). Buffer was removed and samples were snap frozen in liquid nitrogen and stored at -80°C to synchronize experiments. Samples were thawed and treated with nuclear lysis buffer (50mM HEPES pH7.6, 10mM EDTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, ddH2O, 0.1% PIC) and incubated at 4°C for 10 minutes. Chromatin was sheared for 500bp using Branson digital sonifier 250, using 30%, with 30 seconds on 30 seconds 0ff for 5 cycles. Supernatant was snap frozen and stored at -80°C. Immunoprecipitation was carried out using Protein-G SureBeads (BioRad Hurcules, CA, USA). Beads were washed once with 1X PBS prior to blocking with 1X PBS and 0.5% BSA for 20 minutes at 4°C. Samples were precipitated with affinity purified anti-dFOXO antibody (Tatar Lab). Samples were reverse crosslinked at 65°C for 12 hours.
DNA size selection and library prep were done using NEBNext Ultra II DNA library prep kit for Illumina an indexed using NEBNext multiplex oligos for Illumina (Primer set 1) (New England BioLabs, Ipwsich MA). DNA from either ChIP or input samples was mixed with AMPure XP beads (**) to select for a final library size of 320bp. Samples were diluted to a final concentration of 2nM for Illumina sequencing on Illumina 3000 (Illumina, San Diego CA) with single-end 50 bp reads format

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 3000

Description

dFOXO antibody (Tatar Lab)

Data processing

Base calls performed by Illumina HiSeq 3000 Real-Time Analysis software RTA2
Homer Suite-mergePeaks: Collate biological replicates
Galaxy-FastQC: Check for quality
Galaxy-FASTQ Groomer: readability control
Galaxy-Bowtie2: Alignment against dm6 genome
Galaxy-MACS2: Peak calling from alignment reads
BedTools intersect: assign genes to peaks
Genome_build: Aug. 2014 (BDGP R6 + ISO1 MT/dm6)
Supplementary_files_format_and_content: Fold Enrichment values, each group contains two replicates

Submission date

Oct 10, 2018

Last update date

Apr 22, 2021

Contact name

Allison Birnbaum

E-mail(s)

abirnba1@iastate.edu, abirnba1@uab.edu

Organization name

Iowa State University

Street address

3247 Pammel Dr