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Sample GSM1111794

Query DataSets for GSM1111794

Status

Public on Sep 16, 2013

Title

KLE2 Rep2 ChIPSeq

Sample type

SRA

Source name

whole worms

Organism

Caenorhabditis elegans

Characteristics

strain: N2
developmental stage: embryo
matching input: Input_N2_MxEmb_Rep3
antibody: KLE-2 SDQ3942 Rabbit polyclonal is against the antigen MTRNAPPGQESTDLAWLVTPAKDLVENFSIDVLKALAGYLEVIRQESEDTDNQVDAATTYRLFDFQRACRIIQGSCAVYGRKVDHVYELTISVVDLVENK May be available as part of modENCODE antibodies from Novus Biologicals "http://www.novusbio.com/"

Treatment protocol

Embryos were treated with 2% formaldehyde for 30 minutes, washed with M9, and collected by centifugation and freezing at -80C until extract preparation.

Growth protocol

Mixed stage embryos (wild type N2) were isolated by bleaching gravid adults grown in liquid or plates at 20C temperature.

Extracted molecule

genomic DNA

Extraction protocol

Embryos were washed and dounced in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate; 150 mM NaCl). 0.1 sarkosyl was added before sonicating to obtain chromatin fragments of majority between 200-800 bp. 1-2 mg of embryo extract and 3-5 ug of antibody was used per ChIP.
Half of the ChIP DNA were ligated to Illumina or home-made multiplexed adapters and amplified by PCR. Library DNA between 250-500 bp in size was gel purified.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer IIx

Description

KLE2_N2_Mxemb_average.wig
KLE2_N2_Mxemb_peaks.bed

Data processing

Basecalls were performed using CASAVA version 1.8.
ChIP-seq reads were aligned to the WS220 genome assembly using bowtie version 0.12.7 using default parameters for allowing mismatches in the seed (-n option) and suppressing alignments for reads with more than 4 reportable alignments.
Peaks were called using MACS version 1.4.141 with the following setting: input, genome size (-g ce), format (-f BAM), p-value (-p 1e-10 and -p 1e-5)
Genome coverage was estimated using MACS version 1.4.141 with the following setting: genome size (-g ce), format (-f BAM), output in wiggle format (-w), whole genome output (-S), resolution (--space=1)
Coverage per base was normalized to the genome-wide median coverage (excluding the mitochondrial chromosome). Final ChIP enrichment score per base was obtained by subtracting matching input coverage.
Replicates were merged by averaging coverage at each base position. To determine a set of final peaks per subunits, reads from the replicates were combined using the BEDTools utility mergeBam version 2.13.442, and MACS was used to call peaks (see above, p-value -p 1e-10). Only those peaks present in the majority of the individual replicates identified below p-value 1e-5 were included in the final peak set.
To get final peak sets representing condensinI-IDC and condensinII, we used a sliding window across the genome. Each base pair covered by at least two of the three non-SMC subunits (DPY-26, DPY-28, and CAPG-1 for condensinI-IDC, CAPG-2, HCP-6, and KLE-2 for condensinII) was included in the final peak set.
Fragments Per Kilobase of transcript per Million mapped reads (FPKM) were calculated using cufflinks version 2.0.2 using default parameters supplying gene annotations.
Genome_build: WS220
Supplementary_files_format_and_content: Wig files were generated using MACS version 1.4.141, normalized according to the genome-wide mean coverage and the input subtracted. Scores represent the average coverage of each replicate at each base position.
Supplementary_files_format_and_content: Bed files were generated by combining replicate reads and using MACS version 1.4.141 at two different p-values. Only peaks at the more stringend cut-off were inlcuded in the final set that were overlapping with the majority of peaks of each of the replicates at the less stringend p-value.
Supplementary_files_format_and_content: FPKM text file contains the FPKM values for each gene for each replicate.

Submission date

Apr 01, 2013

Last update date

May 15, 2019

Contact name

Sevinc Ercan

Organization name

New York University

Department

Biology

Street address

12 Waverly Place