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Sample GSM1206864 Query DataSets for GSM1206864
Status Public on Aug 11, 2013
Title lola-PJ-GFP_0-12h_embryo_ChIP-seq_ChIP_Rep1
Sample type SRA
Source name lola-PJ-GFP_0-12h_embryo_ChIP-seq_ChIP_Rep1
Organism Drosophila melanogaster
Characteristics strain: lola-RJ-GFP
developmental stage: Embryo 0-12h
genotype: PBac{y[+]-attP-3B}VK00033
transgene: lola-RJ genomic coding region
Extracted molecule genomic DNA
Extraction protocol 1. y; bw cn sp flies are cultivated in cages with apple juice agar plates covered with yeast powder.2. For a 0-12 hours egg laying, new plates are added after a 2hours pre-egglaying at 9pm and flies are allowed to lay eggs for 12 hours.3. Embryos are collected at 8.45 am using a filter mesh and a brush. They are rinsed with EWB and cross-linked at 9am in 1.8% formaldehyde at room temperature for 15 min.
Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample.
DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911). After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer
Description ChIP DNA; Antibody information listed below: official name: GFP ab290;target name: Green Fluorescent Protein;host: Rabbit;antigen: entire GFP sequence;clonal: Polyclonal;purified: Crude;company: Abcam;catalog: ab290;reference: abcam website Jungreis et al. Genome Res. 2011 Dec;21(12):2096-113.;short description: This antibody is reactive against all variants of Aequorea victoria GFP such as S65T-GFP RS-GFP YFP and EGFP.
Data processing Solexa_Alignment protocol. Raw image files were processed following the following Illumina-compatible pipeline:1.  The Raw images were first processed with the Casava  Base-called sequences with confidence metrics were obtained using OLB-1.9.0.  All multiplexed samples were demultiplexed using Casava Finally the BWA program was used to align the sequence reads to the Drosophila melanogaster (v5.32) genome, allowing up to 2 edit distance in the seed region and 3 in the full read length. Tags that aligned to more than one  location were excluded from our analysis for both ChIP sample and the corresponding control sample (input chromatin). Only properly aligned reads with mapping quality more than 30 were kept. MACS_Peak-calling protocol. Peak calls were determined by using Model-based Analysis of ChIP-Seq (MACS) software, version 2 (MACS2). MACS2 was supplied with the following parameters:tagSize = 50mfold = 2genomeSize = 120000000bandwidth = 100pvalue = 1e-5 Processed data are obtained using following parameters: genome version is flybase BDGPv5
Submission date Aug 11, 2013
Last update date May 15, 2019
Contact name DCC modENCODE
Phone 416-673-8579
Organization name Ontario Institute for Cancer Research
Street address MaRS Centre, South Tower, 101 College Street, Suite 800
City Toronto
State/province Ontario
ZIP/Postal code M5G 0A3
Country Canada
Platform ID GPL9058
Series (1)
GSE49772 lola-PJ-GFP_0-12h_embryo_ChIP-seq
BioSample SAMN02315552
SRA SRX467029

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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