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Sample GSM1911034

Query DataSets for GSM1911034

Status

Public on Dec 11, 2015

Title

lem2_cec-4_1

Sample type

SRA

Source name

Early embryo extracts

Organism

Caenorhabditis elegans

Characteristics

developmental stage: early embryo
strain: GW828
genotype/variation: cec-4_delta
chip antibody: anti-LEM-2 (Novus Biologicals, catalog #48540002, lot# T01872A01)

Treatment protocol

Embryos were cross-linked with 2.16% formaldehyde in M9 buffer for 30 minutes at room temperature, washed twice with M9 and once with FA buffer (50mM HEPES-KOH pH7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150mM NaCl)

Growth protocol

Wild-type, met-2 set-25 and cec-4 mutant strains were grown in parallel and in two independent biological replicas. For each strain, 400,000 L1 worms were grown synchronously in 500 ml S-medium containing HB101 E. coli strain, as food source, under continuous agitation (180 rpm) at 20ºC until gravid adults with early embryos were observed (between 60-65 hours depending on strain). Embryonic progeny was harvested using hypochlorite treatment

Extracted molecule

genomic DNA

Extraction protocol

LEM-2 ChIP was performed as described in (Ikegami et al., 2010) with anti-LEM-2 (Novus Biologicals #48540002)
Libraries were prepared from chromatin IP (1.7 -7.4 ng) and input (10 ng) samples using the NEBNext ultra DNA library prep kit for Illumina (NEB # 7370) and the NEBNext Multiplex Oligos for Illumina (NEB # E7335), according to the manufacturer’s recommendations. No size selection was performed during sample preparation and the libraries were indexed and amplified using 15 PCR cycles, using the recommended conditions. After a final cleanup with Agencourt AmPure XP beads (Beckman # A63881), the library size distribution and concentrations were determined using a BioAnalyzer 2100 (Agilent technologies) and Qubit (Invitrogen) instrument, respectively. The final pools were prepared by mixing equimolar amounts of all individual indexed libraries and then sequenced on a HiSeq 2500 (Illumina) in Rapid mode (Paired-End 50).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Data processing

All paired-end ChIP-seq data (2x50bp) were mapped to the c.elegans genome (ce6) with the R package QuasR (http://www.bioconductor.org/packages/3.1/bioc/html/QuasR.html) using the included aligner bowtie [1] allowing only for uniquely mapping read pairs. The command used to perform the alignments was "proj <- qAlign("samples.txt","BSgenome.Celegans.UCSC.ce6")" which instructs bowtie to align using the parameters "--fr -m 1 --best --strata --maxins 500 --phred33-quals". Read density along the genome was calculated by tiling the genome into 200kb windows (non-overlapping) and counting the number of sequence fragments within each window [lem2_win200k_rawCounts.txt]. The command used to create the window count table was qCount(proj,regions,useRead="first"). This instructs QuasR to position each read at the middle of its respective fragment (determined by the two reads) and to only consider the first read (on any strand) for quantification in order to avoid double counting. To compensate for differences in the read depths of the various libraries, we divided each sample by the total number of mapped reads and multiplied by the average library size. Log2 expression levels were calculated after adding a pseudocount of 1 (y=log2(x+1)) [lem2_win200k_normalized.txt].
Genome_build: ce6
Supplementary_files_format_and_content: lem2_win200k_rawCounts.txt
Supplementary_files_format_and_content: lem2_win200k_normalized.txt

Submission date

Oct 19, 2015

Last update date

May 15, 2019

Contact name

Dimos Gaidatzis

E-mail(s)

[email protected]

Organization name

Friedrich Miescher Institute

Street address

Maulbeerstrasse 66