|
| Status |
Public on Jun 07, 2019 |
| Title |
IGN-H3K4me3 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: H3K4me3
antibody: H3K4me3 (61379, 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-H3K4me3 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-H3K4me3 on isolated germ nuclei
IGN-H3K4me3_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 |
SAMN11975695 |
| SRA |
SRX5985674 |
