 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 29, 2012 |
| Title |
Anti-H3K4Me2 ChIP Kdm1a KD |
| Sample type |
SRA |
| |
|
| Source name |
Kdm1a KD AML cells, H3K4Me2 ChIP
|
| Organism |
Mus musculus |
| Characteristics |
background/strain: C57BL/6
genotype/variation: Kdm1a KD
cell type: MLL-AF9 AML cells
chip antibody: H3K4Me2
antibody vendor: Cell Signaling
antibody catalog #: 9725S
antibody lot#: 1
|
| Treatment protocol |
Kdm1a KD or control murine MLL-AF9 AML cells were FACS purified 42 hours following lentiviral infection, with GFP as the selectable marker, and subjected to ChIP-Seq.
|
| Growth protocol |
For experiments involving viral infection of cryopreserved AML cells, cells were thawed, spinoculated as above and cultured overnight with viral supernatant containing 5% X63 supernatant (Karasuyama and Melchers, 1988).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin shearing was performed using a Covaris S2 system (Covaris, Woburn, MA) to 100-300 base pairs, according to manufacturer’s instructions. ChIP-grade antibodies were anti-H3K4Me2, anti-H3K4Me3, anti-H3K9Me2 and anti-H3K9Me3 (all from Cell Signaling). To prepare for next-generation sequencing, libraries were prepared from 2ng immunoprecipitated DNA fragments using the 5500 Series Fragment Library Core Kit and Barcode Adaptors (Applied Biosystems), according to the 5500 SOLiD ‘ALPHA’ ChIP Seq Library Preparation Protocol (Applied Biosystems), with 15 cycles of amplification. Library molarities were determined by Agilent Bioanalyser analysis and then libraries were pooled in equimolar ratios. Emulsion PCR was carried out using the EZBead System (Applied Biosystems) according to the manufacturer’s instructions. An E80 scale emulsion was prepared using 0.4pM of the pooled libraries and the resulting templated beads were modified for 4hrs at 37 C. Next-generation sequencing was carried out on a 5500XL Genetic Analyzer System (Applied Biosystems), according to the manufacturer’s instructions, to generate 50 base pair forward reads.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB 5500xl Genetic Analyzer |
| |
|
| Description |
test_shA4_K4_Me2
|
| Data processing |
Sequencing reads were aligned to the genome (Mus_musculus NCBIM37) using Bowtie (Langmead et al. 2009) with default parameters. Total reads per sample ranged from 2.6 to 6.6 x10^6. Reads that mapped to multiple loci were discarded and the alignments from three technical replicate sets (anti-H3K9Me2 Kdm1a KD) were pooled. Subsequent analysis was performed in R and Bioconductor (Gentleman et al. 2004). Data were then segmented according to annotated transcription start- and end-sites (TSS; TES), as defined by ENSEMBL version 64 and supplied by the annmap database (Yates et al. 2007). Each gene was split into 10 equally spaced bins between the TSS and TES and the average read depth calculated for each region (expressed in reads per kilobase). Similar data were computed for two additional regions 10kB-2.5kB and 2.5kB-0kB upstream of the TSS and the equivalent regions downstream of the TES. In total, 14 regions per gene were considered. In order to explore global changes in histone methylation across the genome of each sample, four sets of genes were considered: MLL-AF9 target genes (n=114), and the remaining protein coding genes (n=15806), stratified into three groups by expression level (0 – 30th percentile, 30th – 70th percentile, 70th – 100th percentile), and the average read depth per bin computed for each set.
BED file: The .BED file contains reads uniquely mapped by Bowtie to the mouse genome for one sample, indicated by the file name. Each line in the .BED file corresponds to one read, showing the mapped location in the mouse genome, i.e. the chromosome, the start coordinate (minus 1), the end coordinate, and the strand.
TXT file: File has 16 columns. On the left is the Ensembl gene ID for the 15920 protein-coding genes analyzed. The next column indicates which of the four analyzed gene sets each gene belongs to (MLL- MLL-AF9 bound; TOP - top 30% most highly expressed non-MLL-AF9 bound genes; MIDDLE - middle 40% expressed non-MLL-AF9 bound genes; BOTTOM - bottom 30% expressed non-MLL-AF9 bound genes). The 14 columns that follow indicate for each gene the ChIP-Seq signal in each of 14 bins, as described in the data processing section. ChIP-Seq signal is defined as mapped reads per kilobase per million reads.
Genome Build:
test_ShA4_K4_2_TS2_11.bed: NCBI Build 37
test_shA4_K4_Me2.txt: ENSEMBL version 64
|
| |
|
| Submission date |
Mar 07, 2012 |
| Last update date |
Mar 07, 2024 |
| Contact name |
Tim C Somervaille |
| E-mail(s) |
tim.somervaille@cruk.manchester.ac.uk
|
| Organization name |
Cancer Research UK Manchester Institute
|
| Lab |
Leukaemia Biology Laboratory
|
| Street address |
Wilmslow Road
|
| City |
Manchester |
| State/province |
Lancashire |
| ZIP/Postal code |
M20 4BX |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL15907 |
| Series (2) |
| GSE36346 |
The histone demethylase KDM1A sustains the oncogenic potential of MLL-AF9 leukemia stem cells (ChIP-Seq data) |
| GSE36348 |
The histone demethylase KDM1A sustains the oncogenic potential of MLL-AF9 leukemia stem cells |
|
| Relations |
| SRA |
SRX129162 |
| BioSample |
SAMN00809348 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM889399_test_ShA4_K4_2_TS2_11.bed.gz |
21.2 Mb |
(ftp)(http) |
BED |
| GSM889399_test_shA4_K4_Me2.txt.gz |
481.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
