|
| Status |
Public on Dec 01, 2013 |
| Title |
CREB with Proteasome Inhibitor |
| Sample type |
SRA |
| |
|
| Source name |
3T3-L1 cells (ATCC #CL-173)
|
| Organism |
Mus musculus |
| Characteristics |
epitope: a.a. 5-24 of human CREB
antibody: 17-600 (Millipore)
molecule: CREB
treatment: lactacystin
|
| Treatment protocol |
Treatment was performed with 25 microM lactacystin or 0.1% DMSO (mock) for three hours.
|
| Growth protocol |
3T3-L1 cells were obtained by ATCC and cultured at undifferentiated state (as fibroblasts) following standard recommendations. 15 Mio cells were processed for an average ChIP-seq run.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After fixation in PBS/1% para-Formaldehyde (37ºC, 10 min), nuclei were isolated by lysis in Triton-X100 buffer and sonicated to ca. 200 bp length. DNA was co-immunoprecipitated with the respective antibodies over night.
Libraries were prepared according to Illumina's instructions (New England Biolabs, Cat. No. E6200). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3 end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
CREB with Proteasome Inhibitor
|
| Data processing |
Alignment: Sequence reads were obtained and mapped to the mouse mm9 genome using the Illumina Genome Analyzer Pipeline.
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/), version 1.4
Genome_build: mm9
Supplementary_files_format_and_content: The bigwigs were created with the following steps: Reads were aligned to the reference genome using bowtie with at most 2 mismatches. The SAM file with those alignments was converted to BAM format and then sorted using samtools, and then the sorted alignments were converted to wig format using a custom python script. Finally, the wigs were converted to bigWigs using wigToBigWig (which can be downloaded from http://hgdownload.cse.ucsc.edu/admin/exe/).
|
| |
|
| Submission date |
Mar 08, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Andre Catic |
| E-mail(s) |
[email protected]
|
| Organization name |
Baylor College of Medicine
|
| Street address |
One Baylor Plaza
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE33821 |
A functional map of DNA-associated proteolysis |
|
| Relations |
| SRA |
SRX247748 |
| BioSample |
SAMN01942211 |
| Named Annotation |
GSM1095380_CREB_Lacta.bigwig |
