|
| Status |
Public on Nov 02, 2015 |
| Title |
Drosophila_larvae_WT_HP1a_ChIP-Seq_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
third instar larvae
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: whole tissue
genotype: wild type
chip antibody: HP1a polyclonal (rabbit)
|
| Treatment protocol |
CDK12 RNAi virgin females were crossed with actin-Gal4 males to drive the ubiquitous knockdown, and the larvae were raised at 18°C. The w1118 strain was crossed with the same Gal4 strain as a wild type control. For total larval chromatin preparation, approximately 3500 third instar larvae were collected, frozen in liquid nitrogen and stored at −80 °C.
|
| Growth protocol |
Larvae were cultured on standard medium (cornmeal/agar/sugar) in 18 degrees.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Using a ceramic mortar and pestle chilled with liquid N2, frozen larvae were ground to a powder and transferred to a homogenizer in ice-cold PBS. At room temperature, the homogenized cell suspensions were cross-linked with 1% formaldehyde for 20 min, quenched with 125 mM glycine for 5 min and filtered through Miracloth. Chromatin pellets were collected and resuspended in ice-cold RIPA buffer, followed by sonicating in a Branson Digital Sonifer with 25% output for 10 min(2s on, 4 s off). For immunoprecipitation, Protein A Sepharose™ Fast Flow (GE Healthcare 17-5280-01) was pre-blocked, 4μl HP1a polyclonal antibody or 3μl H3K9me2 antibody (Ab1220) were used for each immunoprecipitation. The input DNA and the IP DNA were purified with a PCR purification Kit (Qiagen28104 ) and eluted in TE buffer.
Libraries were constructed according to Hiseq 2500 rapid SR50 one flowcell sequencing instructions
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
HP1a polyclonal antibodies are kindly provided by S.C.R. Elgin
|
| Data processing |
The ChIP-seq reads were mapped to Drosophila melanogaster genome (dm3) using the software BWA with default parameters.
After reads alignment, peak calling was performed by software MACS (version 1.4.2).
In addition to the default parameters, some advanced parameters of MACS were set considering that HP1a and H3K9me2 associated with repetitive elements, which are no model, shift size=73 and maximum duplicate tags at the same position=5.
Enrichments of HP1a and H3K9me2 signal were calculated against input.
The occupancy profiles of HP1a and H3K9me2 were recorded into files in wiggle track format (WIG) for analyzing the data in UCSC Genome Browser
Genome_build: dm3
Supplementary_files_format_and_content: wig files, binding profile of HP1a in replicate 2.
|
| |
|
| Submission date |
Aug 28, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Li xia Pan |
| Organization name |
Tsinghua university
|
| Department |
School of medicine
|
| Lab |
C304
|
| Street address |
North Zhongguancun Street
|
| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100084 |
| Country |
China |
| |
|
| Platform ID |
GPL17275 |
| Series (1) |
| GSE63011 |
Heterochromatin remodeling by CDK12 contributes to learning in Drosophila |
|
| Relations |
| BioSample |
SAMN04017558 |
| SRA |
SRX1167419 |
