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Sample GSM1708980 Query DataSets for GSM1708980
Status Public on Dec 07, 2015
Title Ubx_Kc167_Exp1_rep2
Sample type SRA
 
Source name anti-GFP ChIP DNA from Kc167 cells expressing Ubx-GFP (Experiment 1)
Organism Drosophila melanogaster
Characteristics cell line: Kc167
cell line source: Female late embryo derived cell line
antibody: anti-GFP
Treatment protocol To investigate the binding of the different Hox proteins, Kc167 cells were transfected with an inducible expression vector and the GFP-tagged Hox protein transiently expressed before FACS-sorting the Hox-GFP expressing cells. To investigate the binding of Ubx in the presence of Hth, Hth was co-expressed with GFP-tagged Ubx from an inducible bicistronic vector in otherwise Hth non-expressing cells. Detailed protocol is described in associated publication. In order to ensure that the binding profiles for the different samples are as comparable as possible: 1) Cells from the same mother-plate were used for all samples within each of the two experiments (so that the underlying chromatin state available for Hox binding is identical between samples). 2) Cells expressing the same level of GFP-tagged Hox were sorted for all samples across both experiments (so that the nuclear Hox protein concentration available for binding is the same between samples).
Extracted molecule genomic DNA
Extraction protocol Chromatin was prepared from FACS-sorted Hox-GFP expressing cells, and Hox-DNA complexes were immunopurified using anti-GFP antibody. Detailed protocol is described in associated publication.
Samples were processed and sequenced by Source BioScience. The Illumina TruSeq ChIP Sample Preparation Kit was used to generate indexed paired-end sequencing libraries in accordance with the manufacturer’s guide (Rev. A, August 2012) except that no size selection was performed. For the ChIP samples, the entire 10 µl volume of ChIP DNA was used for library preparation and 17 cycles of amplification were performed. For the input sample, 5-10 ng of input DNA were used for library preparation and 12 cycles of amplification were performed. Samples were either sequenced on the Illumina MiSeq (Experiment 1; Ubx, Abd-A, Abd-B and input) or HiSeq 2000 (Experiment 2; Ubx, mutant Ubx and Ubx+Hth) platforms to generate 36 bp or 100 bp reads respectively.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina MiSeq
 
Description Both replicates have been used to call peaks and make the bedGraph file.
peaks and bedGraph files are available on the Ubx replicate 1 record: GSM1708979
Data processing Base call and quality scoring were performed using Real-Time Analysis (RTA) software version 2.0.
Sequencing reads in the raw fastq files were mapped to BDGP release 5 of the Drosophila melanogaster genome (UCSC dm3) using Bowtie1 (version 1.0.1) using default settings for paired-end mapping, a maximum insert size of 1000 bp for valid paired-end alignments (-X 1000) and reporting only uniquely mapped reads (-m 1).
Reads in the output SAM files were processed using Samtools (version 0.1.19) to generate BAM files. Reads from the individual like-replicates were then combined, and filtered using Bedtools (version 2.19.1) to remove the over-represented reads found at the exons of Ubx, Abd-A, Abd-B and Hth; artefacts of the expression vectors used in the transfections.
MACS2 (version 2.1.0.20140616) was used for peak calling using: default settings, the input sample specified as the control file (-c), the -f parameter set to process paired-end BAM files (-f BAMPE), a band width of 200 bp (--bw) and a q-value of 1e-2 or 1e-10 (-q). Binding profiles were generated as fragment pileup tracks in bedGraph format with pileup signal normalized per million reads (-B --SPMR).
Only peaks in euchromatin (chr2L, chr2R, chr3L, chr3R, chr4, chrX and chrM) were used for downstream analyses.
Genome_build: BDGP release 5 (UCSC dm3)
Supplementary_files_format_and_content: BED and bedGraph files were generated using MACS2 (version 2.1.0.20140616) as described above. The BED files contain the called peaks, and the associated peak scores represent −log10(qvalue). The bedGraph files are normalized per million reads and so the different Hox samples are visually comparable when visualized on a genome browser. Both replicates were used in the peak calling process.
 
Submission date Jun 11, 2015
Last update date May 15, 2019
Contact name Robert White
E-mail(s) rw108@cam.ac.uk
Organization name University of Cambridge
Department Physiology, Development and Neuroscience
Street address Downing Street
City Cambridge
ZIP/Postal code CB2 3DY
Country United Kingdom
 
Platform ID GPL16479
Series (1)
GSE69796 Roles of Cofactors and Chromatin Accessibility in Hox Protein Target Specificity
Relations
BioSample SAMN03770077
SRA SRX1056713

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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