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Sample GSM1088966

Query DataSets for GSM1088966

Status

Public on Sep 20, 2013

Title

chico_input_1

Sample type

SRA

Source name

female adult flies

Organism

Drosophila melanogaster

Characteristics

strain: chico heterozygous mutants
age: 15-day-old
chip antibody: none
gender: female
genotype: chico heterozygous mutants
tissue: whole organism

Extracted molecule

genomic DNA

Extraction protocol

About 200-250 adult females (~200 mg) at the age of 15-day-old were pooled for each ChIP sample. Flies were homogenized and cross-linked in 1 X PBS containing 1 % formaldehyde. The fly lysate were sonicated using Branson 450 sonicator to break down the chromatin into a pool of DNA fragment with average size of 500 bp. Immunoprecipitation was performed using Dynal protean A beads (Invitrogen) and affinity purified anti-dFOXO antibody made in our laboratory . Following the wash with LiCl and TE buffer, the DNA-protein complex was eluted from the Dynal beads, reverse-crosslinked and purified.
About 20 ng of ChIP DNA (dFOXO-bound DNA) and input DNA (DNA sample before the immunoprecipitation) were used in library preparation following the methods described in Quail, M.A., et al., Nat Methods. 2008 (5). 1005-1010. Briefly, DNA was end-repaired using an End-It DNA End Repair Kit from Epicentre (# ER0720). The blunt, phosphorylated ends were treated with Klenow (3’->5’ exo-) from NEB (# M0212s) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters. After adapter ligation DNA was PCR amplified with Illumina primers for 18 cycles and library fragments of 150~350 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer IIx

Data processing

Basecalls performed using CASAVA version 1.6
ChIP-seq reads were aligned to the Drosophila reference genome (dmel5.22) using Bowtie
Reads from two replicates were pulled together before peak calling
Peaks were called using PeakSeq with the following setting:
min_threshold = 30 max_threshold = 100 max_gap = 100 number_of_sims = 5 FDR_required = 0.01 bin_size = 10000 max_count = 3 Pf=1 pval_threshold=0.01
Genome_build: dmel5.22
Supplementary_files_format_and_content: candi_final_with_gene.txt files were generated to show the nearest gene next to the candidate peaks

Submission date

Feb 26, 2013

Last update date

May 15, 2019

Contact name

Hua Bai

E-mail(s)

hua_bai@brown.edu

Organization name

Brown University

Department

EEB