|
| Status |
Public on Oct 12, 2016 |
| Title |
Input DNA for ChIPs in YA animals |
| Sample type |
SRA |
| |
|
| Source name |
young adults
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
developmental stage: young adults
tissue: whole animal
strain: AM1061
genotype: unc-119(ed9)III, rmSi1[hsf-1p(4kb)::hsf-1(minigene)::gfp::3'UTR(hsf-1)+Cbrunc-119(+)] II; hsf-1(ok600)I
chip antibody: Input
|
| Treatment protocol |
Worms were either directly collected or subjected to heat shock performed in a water bath pre-heated to 34°C. For heat shock, NGM plates were wrapped with parafilm and submerged for 30 min.
|
| Growth protocol |
Age synchronization was achieved by treatment of alkaline hypochlorite solution and eggs were hatched in M9. L1 larvae were transferred to 10 cm NGM plates seeded with OP50 and grown at 20°C to desired stage.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Worms were collected and washed in cold M9 buffer, crosslinked with 2% formaldehyde at room temperature for 15 min., re-suspended in FA buffer, lysed by douncing in a Kontes 2 ml glass dounce and sonicated in Bioruptor to yield 200 bp–800 bp size DNA fragments. Extracts corresponding to 200 μg of protein from L2 Larvae or 400 μg of protein from young adults was used for each pull-down. For ChIP-seq analysis of HSF-1 and Pol II, 5 μl of antibody against GFP (clontech, polyclonal) or 2.5 μl of antibody against Pol II (Novus) was used in each immunoprecipitation. DNA was pooled from 6 immunoprecipitations for each ChIP-seq experiment.
Libraries were prepared following PrepX DNA library protocol with Appollo 324 system.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
ChIP-seq reads were aligned to the WS220/ce10 assembly of the C. elegans genome using Bowtie 1.1.2 (Langmead 2010) with default settings. Given that several classical heat shock genes (eg. hsp-16.41/hsp-16.2 and F44E5.4/F44E5) are duplicated in C. elegans genome, in addition to uniquely aligned reads, reads with two reportable alignments were also kept. This setting significantly improved occupancy measure and peak calling at the duplicated HSP gene promoters without changing the overall analysis at other loci. Aligned reads were filtered against ModENCODE blacklists (Araya et al. 2014).
The insert size was determined with tools on GeneTrack (Albert et al. 2008) and aligned ChIP-seq reads from both strands were shifted accordingly towards the center.
ChIP-seq data were normalized against the total aligned reads for comparison between experiments. The bedgraph files were generated with bedtools for combined reads from two replicates.
Genome_build: ce10/WS220
Supplementary_files_format_and_content: The bedgraph files were generated with bedtools for combined reads from two replicates and show normalized coverage as reads per million (RPM).
|
| |
|
| Submission date |
May 17, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jian Li |
| E-mail(s) |
jian.li@northwestern.edu
|
| Organization name |
Northwestern University
|
| Department |
Molecular Biosciences
|
| Lab |
Morimoto
|
| Street address |
2205 Tech Drive, Hogan 2-100
|
| City |
Evanston |
| State/province |
IL |
| ZIP/Postal code |
60208 |
| Country |
USA |
| |
|
| Platform ID |
GPL13657 |
| Series (2) |
| GSE81521 |
ChIP-seq of HSF-1 and Pol II in C. elegans larval development and heat shock |
| GSE81523 |
E2F coregulates an essential HSF developmental program that is distinct from the heat-shock response |
|
| Relations |
| BioSample |
SAMN05004799 |
| SRA |
SRX1769999 |
