GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM6588394

Query DataSets for GSM6588394

Status

Public on Dec 31, 2022

Title

exp1, 500mM ChIP

Sample type

SRA

Source name

Bulk whole organism extract

Organism

Caenorhabditis elegans

Characteristics

tissue: Bulk whole organism extract
Sex: pooled male and female
developmental stage: L2
genotype: HW2603 (grh-1(syb616(grh-1::gfp::3xflag))
chip target: GRH-1

Growth protocol

HW2603 worms (grh-1(syb616(grh-1::GFP::3xFLAG)) I) were synchronised by hatching in the absence of food followed by synchronised plating on NA22-containing and peptone rich XL plates at 25°C.

Extracted molecule

genomic DNA

Extraction protocol

5 million animals were collected in PBS + PI + PMSF 1mM at 15 hours and 19 hours after plating (ie 2 timepoints within L2) and popcorn was created from resuspended larvae mix. Popcorn from both time points were pooled and lysate was prepared by grinding the popcorn, crosslinking with 1.1% formaldehyde and sonication with a Diagnode Biorupter Pico. For the chromatin immuno-precipitation, 50 uL of protein G Dynabeads (10003D, ThermoFisher Scientific) were incubated with 5 ug of anti-GFP antibody (ab290, Abcam) for 4 hours rotating at 4°C. 100 ug of chromatin was added, and incubated over night at 4°C rotating. Beads were washed and the elute was treated with proteinase K and RNaseA, and DNA was purified using Zymo ChIP Concentrator Kit.
Libraries were prepared using the ChIP-seq NEB Ultra protocol, and sequenced using the Illumina 50-Cycle Single-End reads protocol on the HiSeq2500.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

Pulldown sample using 500mM salt

Data processing

ChIP-seq SE50 reads were mapped to the C. elegans genome (ce10) using the R function qAlign (splicedAlignment=TRUE, Rbowtie) from the QuasR package (Au et al., 2010; Gaidatzis et al., 2015)
Peaks were identified using the callpeak command from MACS2 (version 2.1.3.3) (Zhang, 2008) on pulldown sample versus corresponding control sample, with thresholding option "-m 2 50", effective genome size option "-g ce", and otherwise default parameters, for each salt concentration independently.
Motifs were identified using HOMER (version 4.11) (Heinz, 2010), using segment size option "-size given", background segment count option "-N 180000", for motifs lengths "-len 8,10,12", autonormalisation "-nlen 3", and parallelisation options.
Putative binding sites were predicted on the non-mitochondrial mappable segments of the ce10 genome (established using function getMappableRegions() from FMI-developped R package swissknife), using the wrapper function findMotifHits() from R package monaLisa (Machlab, 2022), using a minimum score of 6, method "matchPWM", and parallelisation options.
Assembly: ce10
Supplementary files format and content: BED file of MACS2 peaks, filtering out peaks overlapping overmapped genomic regions, and filtering out the 10% least enriched peaks.
Supplementary files format and content: Gzipped BED file of predicted binding sites with scores above 6.

Submission date

Sep 16, 2022

Last update date

Dec 31, 2022

Contact name

Helge Grosshans

E-mail(s)

helge.grosshans@fmi.ch

Organization name

Friedriche Miescher Institute for Biomedical Research (FMI)

Street address

Maulbeerstrasse 66