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Sample GSM3861100 Query DataSets for GSM3861100
Status Public on Jun 07, 2019
Title IGN-H3K27ac ChIP rep2
Sample type SRA
 
Source name Isolated germ nuclei from 2% formaldehyde pre-fixed VC2010 wild type young adult animals
Organism Caenorhabditis elegans
Characteristics developmental stage: young adult (YA)
tissue: Isolated germ nuclei
chipped factor: H3K27ac
antibody: H3K27ac (39685, Active Motif)
strain: VC2010
Extracted molecule genomic DNA
Extraction protocol Growth: VC2010 worms were grown to starvation on NGM plates. Starved L1s were floated on peptone-enriched plates, and grown until gravid at 20˚C. Gravid adults were bleached and synchronized by L1 starvation. L1s were plated to enriched plates and grown until the young adult stage. Animals for nuclei isolation were collected from eighteen enriched plates (~1 million) per ChIP-seq experiment. Young adult animals were harvested at 54-56 hours after plating synchronized L1s when most of the animals had 4-10 embryos. Worms from every six plates (~0.3 million) were collected into one 50 mL conical tube and spun at 3100 rpm for 2 minutes and then washed 3x in M9. Extraction/nuclear preparation: worms were crosslinked in 50 mL 2% formaldehyde for 30 minutes in three 50 mL conical tubes at room temperature. Formaldehyde was quenched by 1 M Tris (pH 7.5) wash. Worms were then washed two more times with M9. Worms were then washed in the same tubes with 10 mL of prechilled Nuclei Purification Buffer. Germ nuclei were isolated following our protocol. Most of the supernatant was removed so that ~20 µL NPB was left. The nuclei were gently pipetted to mix and then flash frozen in liquid nitrogen and stored at -80°C until sonication. IGN samples were sonicated at 2°C in a water bath sonicator (Misonix S-4000). 20% amplitude and 10 sec on/10 sec off pulses were used for a total processing time of 20 minutes, resulting in enrichment for 100-650 bp DNA fragments. 5% of lysate was removed for the input sample and stored at -20°C until the following day to prepare input DNA. 5 μg of anti-H3K27ac was used for each IGN sample. Library construction: The Yale Center for Genome Analysis (YCGA) prepared the library and performed sequencing. The KAPA Hyper Library Preparation kit was used for ChIP sequencing library prep. DNA fragment ends were repaired with T4 DNA Polymerase, and Polynucleotide Kinase and “A” base added using Klenow fragment in a single reaction followed by ligation of custom adapters (IDT) using T4 ligase. Adaptor-ligated DNA fragments were purified and size selected with Agencourt AMPure XP magnetic beads. Adaptor-ligated DNA fragments were amplified by LM-PCR using custom-made primers (IDT). During LM-PCR, unique 10 base indices were inserted at each DNA fragment and amplified products were purified.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer II
 
Description ChIP was performed with anti-H3K27ac on isolated germ nuclei
IGN-H3K27ac_ChIP-input.wig
Data processing ChIP-seq: The raw paired-end ChIP and corresponding input sequencing fastq reads were mapped to ce10 genome by mapping with Bowtie2 (v2.3.2) with default parameters.
ChIP-seq: Wig files were generated by a custom script (https://github.com/guifengwei/glib/blob/master/bam2wig.py),
ChIP-seq: Peaks were called by MACS2 (v2.1.1) with the key parameter (-q 0.001 --nomodel --extsize 150).
RNA-seq: The raw paired-end RNA-seq fastq reads were first mapped to rRNA build by bowtie2 (v2.1.0)
RNA-seq: The remaining unmapped reads were further aligned to ce10 genome by HISAT2 (v2.0.4) with the mode suppressing the unpaired reads
RNA-seq: The gene annotation was downloaded from UCSC Genome Browser, filtered to remove transcripts <50 nt, then was employed to estimate gene expression and call differential gene expression by Cuffdiff program [20] (v2.2.1) under the default parameters between VC2010 and glp-1(q224) strains.
Genome_build: ce10
Supplementary_files_format_and_content: wig or BigWig files were generated as an output
 
Submission date Jun 07, 2019
Last update date Jun 09, 2019
Contact name Valerie Reinke
E-mail(s) valerie.reinke@yale.edu
Phone 203-785-5228
Organization name Yale University School of Medicine
Department Genetics
Lab Reinke lab
Street address 333 Cedar St
City New Haven
State/province CT
ZIP/Postal code 06520
Country USA
 
Platform ID GPL9269
Series (1)
GSE117061 Isolated C. elegans germ nuclei exhibit distinct genomic profiles of histone modification and gene expression
Relations
BioSample SAMN11975703
SRA SRX5985666

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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