|
| Status |
Public on Feb 06, 2019 |
| Title |
RUNX1_ChIP_S00D55H1 |
| Sample type |
SRA |
| |
|
| Source name |
AML samples carrying RUNX1 mutation
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
age: 45-50
subject status: Acute myeloid leukemia
tissue: Venous blood
cell type: Myeloid cell
chip antibody: RUNX1 antibody (Abcam: ab23980)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Purified cells were fixed with 1% formaldehyde (Sigma) at a concentration of approximately 10 million cells/ml. Fixed cell preparations were sonicated using a Diagenode Bioruptor UCD-300 for 3x10 minutes (30 seconds on; 30 seconds off). 67µl of chromatin (1 million cells) was incubated with 229µl dilution buffer, 3µl protease inhibitor cocktail and 0.5-1µg of RUNX1 antibody (Abcam: ab23980) and incubated overnight at 4ºC with rotation. Protein A/G magnetic beads were washed in dilution buffer with 0.15% SDS and 0.1% BSA, added to the chromatin/antibody mix and rotated for 60 minutes at 4°C. Beads were washed with 400µl buffer for 5 minutes at 4°C with five rounds of washes. After washing chromatin was eluted using elution buffer for 20 minutes. Supernatant was collected, 8µl 5M NaCl, 3µl proteinase K were added and samples were incubated for 4 hours at 65°C.Finally samples were purified using Qiagen; Qiaquick MinElute PCR purification Kit and eluted in 20µl EB.
Illumina library preparation was done using the Kapa Hyper Prep Kit. For end repair and A-tailing double stranded DNA was incubated with end repair and A-tailing buffer and enzyme and incubated first for 30 minutes at 20°C and then for 30 minutes at 65°C. Subsequently adapters were ligated by adding 30µl ligation buffer, 10 Kapa l DNA ligase, 5µl diluted adaptor in a total volume of 110µl and incubated for 15 minutes at 15°C. Post-ligation cleanup was performed using Agencourt AMPure XP reagent and products were eluted in 20µl elution buffer. Libraries were amplified by adding 25µl 2x KAPA HiFi Hotstart ReadyMix and 5µl 10x Library Amplification Primer Mix and PCR, 10 cycles. Samples were purified using the QIAquick MinElute PCR purification kit and 300bp fragments selected using E-gel. Correct size selection was confirmed by BioAnalyzer analysis. Sequencing was performed using Illumina NextSeq 500 machines and generated 43bp paired-end reads.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
S00D55H1_RUNX1_HWWMYBGXY_1_11894
|
| Data processing |
Sequenced reads were aligned against the UCSC human reference genome (GRCh37/hg19) with Burrows-Wheeler Aligner (BWA) program with default parameters. The resultant BAM files were subjected to removal of potential PCR and optical duplicates using Picard MarkDuplicates option. The peak calling algorithm MACS2 was used to detect the RUNX1 binding sites at p-value of 10e-6. Peaks overlapping with the consensus excludable ENCODE blacklist and on sex chromosomes were discarded to avoid confounding by repetitive regions and gender-specific bias. All alignment files were extended to the estimated fragment length and scaled to RPKM-normalized read coverage files using deepTools for visualization.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig files were generated using deepTools by scaling read depth to RPKM-normalized coverage
|
| |
|
| Submission date |
Mar 14, 2018 |
| Last update date |
Nov 11, 2021 |
| Contact name |
Joost Martens |
| E-mail(s) |
j.martens@science.ru.nl
|
| Phone |
0243780645
|
| Organization name |
Radboud University
|
| Department |
RIMLS
|
| Lab |
Molecular Biology
|
| Street address |
Geert Grooteplein 28
|
| City |
Nijmegen |
| State/province |
Nederland |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE111821 |
Genome-wide RUNX1 binding landscape in AMLs with RUNX1 mutation |
|
| Relations |
| BioSample |
SAMN08711792 |
| SRA |
SRX3789539 |
