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| Status |
Public on Feb 19, 2015 |
| Title |
SMAD4 MNChIP-seq in cell type HUES64 Smad4_070213_h64 |
| Sample type |
SRA |
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| Source name |
HUES64
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HUES64
cell type: HUES64
chip antibody: SMAD4(Cell Signaling,9515BF,3)
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| Growth protocol |
When human ES cells reached 60-70% confluency on MEFs, the cells were plated as clumps on 6-well plates coated with Matrigel (Life Technologies) in mTeSR1 basal medium (Stem Cell Technologies). We maintained the cells for three days in feeder-free culture and then induced directed differentiation towards mesendoderm (dMS), endoderm (dEN), mesoderm (dME), and ectoderm (dEC) using different media conditions. For mesendoderm and endoderm differentiation cells were cultured for 12 and 120 hours, respectively, in Roswell Park Memorial Institute (RPMI) medium (Life Technologies) supplemented with 100ng/ml Activin A (R&D Systems), 50nM/ml WNT3A (R&D Systems), 0.5% FBS (Hyclone), 200mM GlutaMax (Life Technologies), .2X MEM Non-Essental Amino Acids Solution (Life Technologies), and 55µM b-mercaptoethanol. For the first 24 hours of mesoderm differentiation, cells were cultured in DMEM/F12 medium supplemented with 100ng/ml Activin A (R&D Systems), 10 ng/ml bFGF (Millipore), 100ng/ml BMP4 (R&D Systems), 100ng/ml VEGF (R&D Systems), 0.5% FBS (Hyclone), 200mM GlutaMax (Life Technologies), 0.2X MEM Non-Essental Amino Acids Solution (Life Technologies), and 55µM b-mercaptoethanol. From 24 to 120 hours of mesoderm differentiation, Activin A was removed from the culture. For ectoderm differentiation cells were cultured in DMEM/F12 medium supplemented with 2µM TGFb inhibitor (Tocris, A83-01), 2µM WNT3A inhibitor (Tocris, PNU-74654), 2uM Dorsomorphin BMP inhibitor (Tocris), 15% KOSR (Life Technologies), .2X MEM Non-Essental Amino Acids Solution (Life Technologies), and 55µM b-mercaptoethanol. Media was changed daily.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were grown to a final count of 10 million, resuspended in PBS, and crosslinked in 10% formaldehyde solution for 10 minutes at room temperature. Following quenching with 0.125M glycine and two PBS washes, we isolated nuclei using cell lysis buffer (20 mM Tris-HCl ph8, 85mM KCl, 0.5% NP40). Nuclei were then digested using MNase (Worthington, LS004797) as done in (Henikoff et al., PNAS 2011). Digestion was stopped with 0.05M EGTA and chromatin was aliquoted into 1-2 million cells per ChIP. Antibodies were added and immunoprecipitation was carried out overnight at 4°C as done in1. The next day, protein G beads (Life Technology, 10009D) were added for 2 hours at 4°C to isolate the protein bound DNA and washed twice using Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 150mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 500mM NaCl), LiCl Wash Buffer (0.25M LiCl, 0.5% NP40, 0.5% sodium deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.1,), and TE Buffer pH 8 (10mM Tris-HCl, pH 8, 1mM EDTA pH 8). DNA was eluted twice using 100µL of ChIP Elution Buffer (1% SDS, 0.1M NaHCO3) at 65°C for 15 minutes. Crosslinking was reversed by addition of 32µl reverse crosslinking salt mixture (250 mM Tris-HCl pH 6.5, 62.5 mM EDTA pH 8, 1.25 M NaCl, 5mg/ml Proteinase K) for 5-18 hours at 65°C. DNA was isolated using phenol/chloroform extraction and treated with DNase-free RNase for 30 minutes at 37°C. The whole cell extract (WCE) control was generated using MNase treated material that was then reverse crosslinked and phenol chloroform extracted, skipping the immunoprecipitation and washing steps.
MNChIP-seq library construction was performed as in (Henikoff et al., PNAS 2011) with gel size selection from 140bp to 720bp as described in (Gu et al., 2010). RRBS was performed according to a previously published protocol (Smith et al., 2009) with some optimizations for small cell numbers (Gu et al., 2010).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
SMAD4 MNChIP-seq in cell type HUES64
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| Data processing |
Reads were aligned to the hg19 reference assembly using bwa version 0.5.7 (Li et al., Bioinformatics 2009) with default parameter settings. Subsequently, reads were filtered for duplicates and extended by 200bp. For the bigWig files, extended reads were summed at each base and normalized for sequencing depth by scaling the y-axis to represent cumulative reads per 1 million reads sequenced. DNA methylation calling was performed based on an extended custom software pipeline published previously for RRBS (Gu et al., 2010).
Genome_build: hg19
Supplementary_files_format_and_content: bigWig file containing the normalized occupancy (extended read count) across all genomic coordinates for each MNChIP-seq profile.
Supplementary_files_format_and_content: BED format file which contains the peak locations.
Supplementary_files_format_and_content: bigBed file containing all seen CpGs within this library. The number of methylated reads/number of total reads is listed in the score column
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| Submission date |
Sep 16, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Alexander Tsankov |
| E-mail(s) |
atsankov@alum.mit.edu
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| Organization name |
Harvard University
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| Street address |
7 Divinity Drive
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE61475 |
Transcription factor binding dynamics during human ES cell differentiation |
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| Relations |
| BioSample |
SAMN03069992 |
| SRA |
SRX702098 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1505757_Smad4_070213_h64.bed.peak.txt.gz |
257.6 Kb |
(ftp)(http) |
TXT |
| GSM1505757_Smad4_070213_h64.bw |
236.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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