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| Status |
Public on Oct 12, 2017 |
| Title |
ESC (alternative genotype) H3K9me2 ChIP-seq rep1 |
| Sample type |
SRA |
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| Source name |
Embryonic Stem Cell
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| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic Stem Cell
genotype: CMV-creERT; Hdac3f/f
chip antibody: H3K9me2: ab1220 [Abcam]
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| Treatment protocol |
For differentiation of wild-type cells related to LaminB and H3K9me2 ChIP-seq experiments, the protocol was slightly modified: mESCs were cultured in serum-free, feeder-free 2i media (50% DMD-F12 (Invitrogen); 50% Neurobasal medium (Invitrogen); 0.5 x N2 supplement (Invitrogen); 0.5 x B27 supplement (Invitrogen); 0.05% BSA (Invitrogen); L-Glutamine (Invitrogen); Penicilin/Streptomycin (Invitrogen) and 150 uM ß-mercaptoethanol (Sigma); 20 ng/ml LIF (eBioscience), 3 µM CHIR (Stemgent) and 1 µM PD325901 (Stemgent). ESCs were differentiated as EBs to the cardiomyocyte lineage. EBs were generated at day 0 of differentiation in SF-D media (75% IMDM (Invitrogen); 25% Ham’s F12 (Invitrogen); 0.5 x N2 supplement (Invitrogen); 0.5 x B27 supplement (Invitrogen); 0.05% BSA (Invitrogen); L-Glutamine (Invitrogen); Penicillin/Streptomycin (Invitrogen) and 150 µM beta-mercaptoethanol (Sigma) with ascorbic acid (0.5 mM) and monothioglycerol (0.4 mM). On day 2, EBs were collected, dissociated, and reaggregated in SF-D media with VEGF (5 ng/ml, R&D Systems), Activin A (8 ng/ml, R&D Systems), and BMP4 (1 ng/ml, R&D Systems) to induce cardiac mesoderm formation. At day 4 EBs were collected, washed (with IMDM), and cultured in StemPro (Invitrogen) with VEGF (5 ng/ml, R&D Systems), bFGF (10 ng/ml, R&D SYstems), and XAV (10 µM, Stemgent) to induce cardiomyocyte differentiation. Subsequent media changes were done in the same media without XAV, and cells were collected on day 10 of differentiation.
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| Growth protocol |
Embryonic stem cells (ESCs) were derived from CMV-creERT; Hdac3f/f mice as previously described {Nagy, 2002 #108}. Briefly, blastocysts were collected at E3.5 and cultured on STO feeder cells in standard ESC media + leukemia inhibitory factor (LIF) + 50µM MEK1 inhibitor (Cell Signaling #9900) for 7 days until the blastocyst hatches and forms colony. Individual colonies were subcultured for about 1 week when MEK1 inhibitor is removed and cells passaged as a normal ESC line. Cardiac differentiation from these cells was adapted from published protocols {Christoforou, 2008 #9;Kattman, 2011 #18}. Briefly, ESCs were cultured and maintained on a feeder layer of mitotically inactivated MEFs in DMEM with 15% FBS (Fisher Scientific # SH3007003) and ESGRO LIF. Differentiation through hanging droplets method was initiated following ESC dissociation and suspension at 5 x 104 cells/ml in DMEM with 10% FBS (Atlanta Biologicals #S11550) without LIF in 20 µl drops. Two days after droplets formation, embryoid bodies (EBs) were transferred in suspension onto poly-HEMA coated dishes. After another two days, EBs were plated on gelatin coated dishes in cardiac differentiation media (StemPro-34 SF medium (Invitrogen #10639-011) supplemented with 5ng/ml VEGF (R&D systems), 10ng/ml bFGF (R&D systems), 12.5ng/ml FGF10 (R&D systems), 2.5µM XAV939 (Cayman Chemical #13596), 1mM Ascorbic Acid (Sigma #A4403) and 2mM Glutamax (Invitrogen #35050-061)). Beating cells were visible within 24-48 hours. Hdac3 knockout in CMV-creERT; Hdac3f/f ES cells were performed using 4-Hydroxytamoxifen (Sigma #T176) dissolved in 100% ethanol. Cells were treated with 1 µg/ml tamoxifen for 24 hour to delete Hdac3.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Murine ESCs and ESC-derived CMs were crosslinked in culture by addition of methanol-free formaldehyde (ThermoFisher; final 1% v/v) and incubated at room temperature for 10 minutes with gentle rotation. Crosslinking was quenched by addition of glycine (final 125mM) and incubated at room temperature for 5 minutes with gentle rotation. Media was discarded and replaced with PBS; cells were scraped and transferred to conical tubes and pelleted by centrifugation (250xg, 5 minutes at room temperature). Supernatant was removed, and pellets were flash frozen on dry ice and stored at -80C. Six hours before lysing cells, 30uL protein G magnetic beads (per ChIP sample) were washed 3 times in blocking buffer (0.5% BSA in PBS); beads were resuspended in 250uL blocking buffer and 2ug antibody (Lamin B: sc6217 [Santa Cruz]; H3K9me2: ab1220 [Abcam]) and rotated at 4C for at least 6 hours. Crude nuclei were isolated from frozen crosslinked cells as follows: cell pellet (from 10cm plate) was resuspended in 10mL cold Lysis Buffer 1 (50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP‐40, 0.25% Triton X‐100, and protease inhibitors), and rotated at 4C for 10 minutes, followed by centrifugation (250xg, 5 minutes at room temperature). Supernatant was discarded and the pellet was resuspended in 10mL cold Lysis Buffer 2 (10mM Tris‐HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, and protease inhibitors), and rotated at room temperature for 10 minutes, followed by centrifugation (250xg, 5 minutes at room temperature). Supernatant was discarded and nuclei were resuspended/lysed in 1mL cold Lysis Buffer 3 (10mM Tris‐HCl, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na‐Deoxycholate, and protease inhibitors) and transferred to pre-chilled 1mL Covaris AFA tubes (Covaris) Samples were sonicated using a Covaris S220 sonicator (high cell chromatin shearing for 15 minutes; Covaris). Lysates were transferred to tubes and Triton X-100 was added (final 1%) followed by centrifugation (top speed, 10 minutes at 4C in microcentrifuge). Supernatant was transferred to a new tube; protein concentration was measured by Bradford assay. Antibody-conjugated beads were washed 3 times in blocking buffer, resuspended in 50uL blocking buffer and added to 500ug input protein for overnight incubation with rotation at 4C. 50ug lysate was aliquoted and stored at -20C for input. On day 2, beads were washed 5 times in 1mL RIPA buffer (50mM HEPES‐KOH pH 7.5, 500mM LiCl, 1mM EDTA, 1% NP‐40, 0.7% Na‐Deoxycholate) with 2-minute incubation at room temperature with rotation for each wash. Beads were washed in 1mL final wash buffer (1xTE, 50mM NaCl) for 2 minutes with rotation at room temperature before final resuspension in 210uL elution buffer (50mM Tris‐HCl pH 8.0, 10mM EDTA, 1% SDS). To elute, beads were incubated with agitation at 65C for 30 minutes. 200uL eluate was removed to a fresh tube, and all samples (ChIP and reserved inputs) were reverse-crosslinked overnight at 65C with agitation for a minimum of 12 hours, but not more than 18 hours. 200uL 1xTE was added to reverse crosslinked DNA to dilute SDS, and samples were RNaseA treated (final 0.2mg/mL RNase; 37C for 2 hours) and Proteinase K (final 0.2mg/mL Proteinase K; 55C for 2 hours) before phenol:chloroform extraction and resuspension in 10mM Tris-HCl pH 8.0.
ChIP and input DNA was quantified by Qubit (ThermoFisher) before library preparation using the NEBNext Ultra II DNA library prep kit (NEB). Samples were indexed for multiplex sequencing. Library quality was analyzed by BioAnalyzer (Agilent Genomics) and quantified using qPCR (Kapa Biosystems). Libraries were pooled for multiplex sequencing, requantified, and sequenced on the Illumina NextSeq500 platform (vII; 75bp single end sequencing; Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
mESC_HDAC3-ERCre_MinusTamoxSet1_H3K9me2
mESC_H3K9me2.bw
mESC_H3K9me2_g_10_bin_auto_fdr_5_peaks.bed
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| Data processing |
Fastq files were aligned to the mm9 reference genome using STAR with the following parameters --alignIntronMax 1 --alignEndsType EndToEnd
Duplicate reads were marked and removed using Picard Tools and Bamtools
Replicates were merged and downsampled using PicardTools
Peaks were called using EDD using the following parameters '--bin-size auto --fdr 5 -g 10 '
Coverage tracks were created using DeepTools BamCompare
Genome_build: mm9
Supplementary_files_format_and_content: Input normalized coverage wiggle files and EDD peak bedfiles
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| Submission date |
Apr 17, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael Patrick Morley |
| E-mail(s) |
mmorley@pennmedicine.upenn.edu
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| Phone |
215-898-2026
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| Organization name |
Perelman School of Medicine at the University of Pennsylvania
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| Department |
Penn Cardiovascular Institute
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| Street address |
3400 Civic Center Blvd, Bldg 421
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE97877 |
LaminB and H3K9me2 ChIP-seq During Mouse Cardiogenesis |
| GSE97878 |
Genome-nuclear lamina interactions regulate progenitor cell lineage restriction during cardiogenesis |
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| Relations |
| BioSample |
SAMN06759774 |
| SRA |
SRX2741882 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2579541_mESC_HDAC3-ERCre_MinusTamoxSet1_H3K9me2_filter.bw |
110.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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