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Sample GSM2339594

Query DataSets for GSM2339594

Status

Public on May 09, 2017

Title

SDC2 N2 MxEmb rep1 ChIP-seq

Sample type

SRA

Source name

whole worms

Organism

Caenorhabditis elegans

Characteristics

strain: N2
developmental stage: mixed embryo
antibody target: SDC-2
antibody (vendor, catalog#, lot#): SDI, SDQ3146, G3048-089
antibody antigen: 1749-1848aa
antibody (host, clone): Rabbit, polyclonal
matching input: EE6 input N2 MxEmb ChIP-seq

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

Embryos were isolated by bleaching gravid adults grown on plates at 20C.

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 II

Description

SDC2_N2_MxEmb_MACS141_e5_average.bw
SDC2_N2_MxEmb_MACS141_e10_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 1.0.1 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 1.4.2 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 1.4.2 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, 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.
RNA-seq reads were aligned to the WS220 genome assembly using tophat version 2.0.12 using default parameters, specifying first strand alignment.
Resultant RNA-seq bam files were converted to sam files using samtools version 1.2. Sam files were used to generate count data using htseq-cout version 0.6.1.
Genome_build: WS220
Supplementary_files_format_and_content: BigWig files were generated by converting wiggle files using the UCSC wigToBigWig utility. Wiggle files were generated using MACS version 1.4.2, 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 1.4.2 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: Counts files were generated using Htseq-counts version 0.6.1.

Submission date

Oct 07, 2016

Last update date

May 15, 2019

Contact name

Sarah Elizabeth Albritton

E-mail(s)

sarahea283@gmail.com

Organization name

New York University

Department

Biology