|
| Status |
Public on Nov 04, 2020 |
| Title |
H3k4me3_2 |
| Sample type |
SRA |
| |
|
| Source name |
human cardiac 2D cultures
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: 2D pluripotent stem cell derived cardiac cultures
antibody: anti-H3K4me3 (Active Motif; 39159)
|
| Treatment protocol |
2D cardiac cells were treated for 24 hours with 5 µm CHIR.
|
| Growth protocol |
Briefly, HES3 and H9 hESCs (WiCell) or hiPSC (Sendai-virus reprogrammed CD34+ cells, ATCC) were maintained as TypLE (ThermoFisher Scientific) passaged cultures using mTeSR-1 (Stem Cell Technologies)/Matrigel (Millipore). hPSCs were seeded at 2´104 cells/cm2 in Matrigel-coated flasks and cultured for 4 days using mTeSR-1. They were then differentiated into cardiac mesoderm using RPMI B27- medium (RPMI1640 GlutaMAX+ 2% B27 supplement without insulin, 200 μM L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate (Sigma) and 1% Penicillin/Streptomycin (all ThermoFisher Scientific) containing 5 ng/ml BMP-4 (RnD Systems), 9 ng/ml Activin A (RnD Systems), 5 ng/ml FGF-2 (RnD Systems) and 1 μM CHIR99021 (Stem Cell Technologies) with daily medium exchange for 3 days. Subsequently, they were specified into a cardiomyocyte/stromal cell mixture using RPMI B27- containing 5 μM IWP-4 (Stem Cell Technologies) followed by another 7 days of RPMI B27+ (RPMI1640 GlutaMAX + 2% B27 supplement with insulin, 200 μM L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate and 1% Penicillin/Streptomycin) with medium exchange every 2-3 days. The differentiated cells were then cultured in RPMI B27+ until digestion at 15 days using 0.2% collagenase type I (Sigma) in 20% fetal bovine serum (FBS) in PBS (with Ca2+ and Mg2+) for 60 min at 37°C, followed by 0.25% trypsin-EDTA for 10 min. The cells were filtered using a 100 mm mesh cell strainer (BD Biosciences), centrifuged at 300 x g for 3 min, and resuspended at the required density in CTRL medium: α-MEM GlutaMAX, 10% FBS, 200 μM L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate and 1% Penicillin/Streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were washed 2x with ice cold PBS and then scraped in 1mL PBS into a 1.5mL Eppendorf tube. Fixed cells were spun (200xg, 10 minutes) and treated with lysis buffer according to the MAGnify Chromatin Immunoprecipitation kit protocol (150 μL/3 million cells assuming 15x106 cells/flask). Chromatin was sheared using a Bioruptor UCD2000 sonicator with the following settings: 12x cycles of 30 seconds sonication with 90 seconds cooling. Chromatin was sonicated in 1.5 mL eppendorf tubes, and the volume of cell lysate in each tube during sonication was kept at 150 μL. Sheared chromatin in lysis buffer was stored at -80°C prior to ChIP. ChIP reactions were carried out with MAGnify Chromatin Immunoprecipitation System (ThermoFisher) reagents as per manufacturer’s instructions. Antibodies used in ChIP reactions were: 1 μg anti-TCF4 (Santa Cruz: sc-8631 X), 3 μL anti-H3K4me3 (Active Motif; 39159), 5 μg anti-H3K27ac (Active Motif; 39133).
ChIP-seq Libraries were created with TruSeq ChIP Library Preparation Kit (Illumina) with DNA size selected and quality controlled by Pippin Prep size selection (Sage Science). Libraries were read with HiSeq SR Cluster v4 kit (Illumina) on a HiSeq 2500 sequencer with 50-bp single-end reads.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
H3K4me3_consensus.bed
|
| Data processing |
FASTQ files were quality trimmed with Trimmomatic (v 0.36.6)
Reads were then mapped with Bowtie2 (v 2.3.4.1) to GRCh37/hg19
Peaks were called with the callpeak function of MACS2 (v 2.1.1.0) with default parameters (q-value < 0.01). For H3K4me3 and H3K27ac files, broadpeaks were called using broad subcommand with --broad.cutoff= 0.1.
Replicate peaks were intersected using the intersect function of GenomicRanges (v1.34.0)
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: Processed files are text files indicating peaks in bed format.
|
| |
|
| Submission date |
May 26, 2020 |
| Last update date |
Nov 04, 2020 |
| Contact name |
James Hudson |
| E-mail(s) |
James.Hudson@QIMRBerghofer.edu.au
|
| Organization name |
QIMR Berghofer Medical Research Institute
|
| Lab |
Cardiac Bioengineering Laboratory
|
| Street address |
300 Herston Rd
|
| City |
Brisbane |
| State/province |
QLD |
| ZIP/Postal code |
4006 |
| Country |
Australia |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE150521 |
β-catenin drives distinct transcriptional networks in proliferative and non-proliferative cardiomyocytes |
|
| Relations |
| BioSample |
SAMN15017567 |
| SRA |
SRX8401250 |
