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Sample GSM3044960 Query DataSets for GSM3044960
Status Public on Feb 06, 2019
Title RUNX1_ChIP_d31_DOX_iPSC
Sample type SRA
 
Source name iPSC
Organism Homo sapiens
Characteristics mutation status: DOX-induced RUNX1mut
harvest time: day 31
cell type: iPSC
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
 
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 15, 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 (2)
GSE111917 RUNX1 mutations lead to a myeloid differentiation block by altering the RUNX1 transcriptional program (ChIP-Seq)
GSE111919 RUNX1 mutations lead to a myeloid differentiation block by altering the RUNX1 transcriptional program
Relations
BioSample SAMN08720332
SRA SRX3800484

Supplementary file Size Download File type/resource
GSM3044960_RUNX1mt-d31-dox-12985.bw 345.7 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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