|
| Status |
Public on Jan 10, 2012 |
| Title |
T-ALL mnase input replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
T-ALL mnase input
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Notch1-IC over-expressing tumors (T-ALL)
chip antibody: none (input)
|
| Treatment protocol |
We fixed the cells with 1% formaldehyde for 10 min at 25oC and mononucleosomal particles were generated using micrococcal nuclease (from USB). We stopped the reaction with the addition of EDTA (20 mM), and the nuclei were lysed using the ‘Nuclei lysis buffer’ (50 mM Tris-HCl pH 8.0), 10 mM EDTA (pH 8.0) and 1% SDS) followed by sonication (2.5 min in total) using the bioruptor from Diagenode and addition of nine volumes of ‘IP dilution’ buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA (pH 8.0), 16.7 mM Tris-HCl pH 8.0 and 167 mM NaCl) and addition of magnetic Dynal beads (preclearing of chromatin). For the ChIP of Notch1, we fixed the cells with 1% formaldehyde for 10 min at 25oC and lysed them using the ‘cell lysis’ buffer (5 mM HEPES pH 8.0, 85 mM KCl and 0.5% NP-40). We lysed the nuclei using the Nuclei lysis buffer (50 mM Tris-HCl pH 8.0), 10 mM EDTA (pH 8.0) and 1% SDS) followed by sonication (40 min in total) using the bioruptor from Diagenode and addition of nine volumes of IP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA pH 8.0, 16.7 mM Tris-HCl pH8.0 and 167 mM NaCl) and addition of magnetic Dynal beads (preclearing of chromatin). We used standard ChIP procedures adapted to our cell numbers (~1–5 ´ 106 cells) for ChIP.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq libraries were generated using standard Illumina kit and protocol. We started preparing libraries using 5 to 10ngs of DNA. We sequenced 6.5 pmoles of library preparation using GAII illumina sequencer. We performed 36bp (34bp after processing) single-end run.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
ChIP-Seq analysis for input in T-ALL cells replicate 2
|
| Data processing |
We aligned the sequenced reads to the genome (mouse assembly NCBIM37/mm9) using Burrows-Wheeler Alignment (BWA) with maximum two mismatches. The ChIP-seq reads were validated regarding the reproducibility of the replicates, and the sensitivity and specificity of each activating and repressive histone mark in classifying genes of high versus low expression and average read profiles of genes of high expression versus genes of low expression were constructed in TSSs flanked by 10 kb.
We determined epigenetic changes between the double-positive and T-ALL samples by evaluating sliding windows across the genome using an approach adapted from PeakSeq. We performed all ChIP-seq read data manipulations using GenomicTools (http://code.google.com/p/ibm-cbc-genomic-tools/), and the algorithm for identifying epigenetic changes in T-ALL compared to double-positive was implemented in R language. Notch1 ChIP-seq peaks in T-ALL were determined using MACS.
|
| |
|
| Submission date |
Jan 09, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Aristotelis Tsirigos |
| E-mail(s) |
[email protected]
|
| Organization name |
IBM Research
|
| Department |
Computational Biology Center
|
| Lab |
Computational Genomics
|
| Street address |
1101 Kitchawan Road, Route 134
|
| City |
Yorktown Heights |
| State/province |
NY |
| ZIP/Postal code |
10598 |
| Country |
USA |
| |
|
| Platform ID |
GPL9250 |
| Series (1) |
| GSE34954 |
Notch1-driven epigenetic changes in a mouse model of T cell Acute Lymphoblastic Leukemia (T-ALL) |
|
| Relations |
| SRA |
SRX114991 |
| BioSample |
SAMN00771308 |
