|
| Status |
Public on Dec 05, 2016 |
| Title |
ago1D 0h, input for sample 21, rep1 (ChIP-Seq) |
| Sample type |
SRA |
| |
|
| Source name |
Cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: ago1D (975 h+ ago1D::NatMX6)
treatment: control
time: 0h after -N
ip antibody: none
|
| Growth protocol |
Cells grown in EMM 225ug/ml adenine/histidie/leucine/lysine/uracil (OD~1) at 32C, and were washed twice with EMM -N. Cells were resuspended in EMM -N and incubated at 32C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Equal number of cells at each time point was cross-linked with 1% formaldehyde at room temperature for 15min. Crosslinking was quenched with 0.125M glycine for 5min and the cells were washed 3 times with cold TBS. Cell pellets (about 2x109 cells) were resuspended in 400μl lysis buffer (20mM Hepes, 0.5M NaCl, 1mM EDTA, 1% Triton-X, 0.1% Na-Deoxycholate, 0.1% SDS, 1mM PMSF and 5mM Benzamidine) and lysed with glass beads. Whole cell extracts (WCEs) were sonicated for 10min using the Q-Sonica waterbath sonicator with final energy output of roughly 16000J producing chromatin 100-400bps in size. 2-4ug of antibody (abcam 1220 for H3K9me2 and active motif 61013 for H3K9me3) plus 10μl Protein A Dynabeads were added to each extract and incubated for 3 hours at 4 deg. Beads were then washed and eluted with TE plus 0.67% SDS at 65ºC for 15min. Samples were incubated at 65 ºC for 8 hours to reverse the crosslinks and then treated with RNaseA and proteinase K to remove RNA and proteins, respectively. Finally, DNA fragments were isolated by Phenol-Chloroform extraction followed by and ethanol precipitation.
Libraries were made with NEBNext Ultra DNA Library rep Kit for Illumina (NEB E7370L)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Sample 47
|
| Data processing |
Map to S pombe genome with Bowtie2 2.2.7
Identify H3K9me2 peaks by MACS2 2.1 (--nomodel --extsize 200 --keep-dup auto)
Identify differentially bound peaks by DiffBind 1.16.3 which uses DESeq2 using replicate 1/3/4/5/6 of wt 0h and 24h
Genome_build: S. pombe ASM294v2.22
Supplementary_files_format_and_content: bedgraph
Supplementary_files_format_and_content: Text. List of differentially bound peaks of H3K9me2 between wt 0h and wt 24h
|
| |
|
| Submission date |
Oct 25, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard Joh |
| E-mail(s) |
rich.i.joh@gmail.com
|
| Organization name |
Mass General Hospital
|
| Department |
Center for Cancer Research
|
| Street address |
149 13th St
|
| City |
Charlestown |
| State/province |
MA |
| ZIP/Postal code |
02129 |
| Country |
USA |
| |
|
| Platform ID |
GPL20584 |
| Series (2) |
| GSE89142 |
Survival in quiescence requires the euchromatic deployment of Clr4/SUV39H by Argonaute-associated small RNAs [ChIP-Seq] |
| GSE89151 |
Survival in quiescence requires the euchromatic deployment of Clr4/SUV39H by Argonaute-associated small RNAs |
|
| Relations |
| BioSample |
SAMN05938845 |
| SRA |
SRX2267816 |
