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| Status |
Public on Feb 26, 2019 |
| Title |
Input_6day_dox_rep1 |
| Sample type |
SRA |
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| Source name |
differentiating mES cells
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| Organism |
Mus musculus |
| Characteristics |
tissue: differentiating mES cells
differentiation protocol: serum
time: day9 of differentiation
purification: PDGFRα+FLK1- sorted
doxycycline-treatment: 6-day
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| Growth protocol |
Mouse ES cell differentiation was achieved by diluting 40000 cells/ml in EB differentiation medium and incubation in an orbital shaker at 80 RPM. EB differentiation medium: IMDM supplemented with 15% FBS , 1% Penicillin/Streptomycin, 2mM Glutamax, 50 μg/ml Ascorbic acid, 4.5 mM Monothioglycerol. For Serum-free differentiation, FBS was replaced with an equivalent amount of Knock-outTM Serum Replacement (Invitrogen). Trangene induction was achieved by adding doxycycline (Sigma-Aldrich) to day 3 EBs cultures (final concentration 1µg/ml), and then maintained throughout the differentiation protocol by replacing the media (including dox) every two days. At day 5, EBs were disgregated and single cells were incubated for 20 minutes with PDGFRα-PE and FLK1-APC conjugated antibodies. PDGFRα+FLK1- cells were sorted using FACSAriaII (BD biosciences) and replated on gelatin coated dishes using serum- or serum-free EB differentiation media supplemented with 1µg/ml doxycycline and 10ng/ml mouse basic-FGF.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq: D4 EBs (1-day induced and control Pax3 mouse cells) were trypsin-treated at 37°C for 1 minute with gentle shacking and reaction was inhibited by adding 10%FBS/PBS. 1-day induced Pax3 NIH3T3 cells and 6-day induced Pax3 mouse cells growing in monolayer were harvested by incubation with trypsin for 1-2 minutes. Human differentiating PAX3-inducible cells were collected at day 6 of differentiation (following 24h dox-mediated induction) by incubation with trypsin for 1-2 minutes. Single cells were washed once with PBS, resuspended in 10%FBS/PBS and supplemented with formaldehyde (final concentration 1%) for crosslinking of protein-DNA complexes (10 minutes at RT) followed by quenching with glycine and staining with PDGFRα-PE antibody. PDGFRα+ cells were sorted using FACSAriaII, snap-frozen in liquid nitrogen and stored at -80°C if not processed immediately. Cell pellets were incubated in lysis buffer LB1 supplemented with protease inhibitors (50mM HEPES KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100 + Complete-mini - Roche) for 10 minutes at +4°C followed by incubation in buffer LB2 supplemented with protease inhibitors (10mM TRIS HCl pH 8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA + Complete-mini - Roche) for 10 minutes at +4°C. Cell pellet was then resuspended in LB3 supplemented with protease inhibitors (10mM TRIS HCl pH 8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-lauroylsarcosine + Complete-mini - Roche) and then sonicated. Cells were sonicated with a Branson sonicator at 18% power for 1 minute with intervals of 10 sec ON-10 sec OFF to achieve an average chromatin size of 300bp. After shearing, samples were centrifuged for 10 minutes at 16000g and snap frozen in liquid nitrogen if not processed immediately. For Pax3 ChIP, 25-40µg of chromatin (diluted to 500µl) were precleared for 4h at 4°C with 20µl of BSA-blocked Protein A (or Protein G)-conjugated sepharose beads (GE healthcare). Samples were supplemented with 1/10 volume of 10% Triton X-100 and incubated overnight with anti-Pax3 (Santa Cruz Biotechnology sc-34926) antibody. Immune complexes were recovered by incubation with 20µl of BSA-blocked Protein G-conjugated sepharose beads for 4h at 4°C and then washed 5 times with RIPA wash buffer (50mM HEPES KOH pH7.5, 500mM LiCl, 1mM EDTA, 1% NP40, 0.25% Triton X-100, 0.7% Sodium Deoxycholate) and one time with TEN buffer (10mM TRIS HCl pH 8, 1mM EDTA, 50mM NaCl). Immunoprecipitated chromatin was recovered by incubating beads with 200µl of Elution buffer (50mM TRIS HCl pH 8, 10mM EDTA, 1% Sodium Dodecyl Sulfate) for 20 minutes at 65°C. Chromatin from IP and Input (equivalent to 1% of starting material) was reverse crosslinked overnight at 65°C, then diluted 1:1 with TE (10mM TRIS HCl pH 8, 1mM EDTA) supplemented with 4µl of RNaseA 20mg/ml and incubated for 2 hours at 37°C followed by Proteinase K treatment (4µl of 20mg/ml stock for each sample) for 30 minutes at 55°C. DNA was purified by Phenol-chloroform-isoamyl alcol extraction (twice) followed by chloroform extraction, then supplemented with 1/10 of volume of 3M Sodium Acetate pH 5 and 1.5µl of Glycogen and precipitated with 2 volumes of 100% Ethanol at -80°C for >1 hour. Followed 30 minutes centrifuge at 16000g, pellet were washed with 75% ethanol, air dried and dissolved in 45µl H2O.
ChIP-seq libraries were generated following a gel-free protocol using AMPure XP beads (Beckman Coulter) for all the purification and size selection steps. 10ng or less of DNA were end repaired using End-it DNA end repair (Epicentre), then A-tailed using Klenow Fragment (3'→5' exo- NEB) followed by adapter-barcode ligation using T4 DNA ligase (Enzymatics). Illumina compatible adapter-barcodes were purchased from BIOO scientific. After ligation, DNAs were negatively size selected using 0.5x Ampure XP beads and unbound DNAs were positively size selected by adding 0.4x Ampure XP beads (this step allows for retention of DNA fragments ranging 200-500bp). Libraries were amplified using Phusion High Fidelity PCR master mix 2x (NEB) with a 16 cycles program. Libraries from iPax3 NIH3T3 cells were generated using the NEBNext DNA library prep kit (NEB).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
processed data file:
mouse_Pax3_1day_ChIP_peaks.bed
Input_6day_plusdox_01
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| Data processing |
ChIP-seq analysis. Each sample’s reads were aligned to the mouse (mm10) or human (hg38) genome using Bowtie2 followed by removal of PCR duplicates using Samtools. Peak calling was performed using MACS with the following parameters: --bw 300 -p 1e-3. Similar results were obtained by performing peak calling using QESEQ with the following parameters: -s 100 -c 15 -p 0.01. To identify the list of high confidence mouse Pax3 peaks we performed 3 independent Pax3 ChIP-seq experiments and, using the bedtools intersect function, only common regions between 2 experiments were further considered. In addition, the Pax3 peak list was filtered (intersect –v option) for sites overlapping to peaks detected in the uninduced control ChIP-seq and in the mouse (or human) ChIP-seq black-list. Density maps were generated with SeqMiner using the enrichment file enr.sgr produced by QESEQ. Bigwig files for visualization on IGV were generated by converting the wig files obtained from MACS using the Kent tool Wig-to-BigWig.
Genome_build: mm10
Supplementary_files_format_and_content: ChIP-seq: processed files represent list of Pax3 peaks obtained by removing Encode blacklist and peaks detected in non-induced samples or input
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| Submission date |
Jan 16, 2019 |
| Last update date |
Feb 26, 2019 |
| Contact name |
Alessandro Magli |
| E-mail(s) |
alemagli@gmail.com
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| Organization name |
University of Minnesota
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| Department |
Medicine
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| Street address |
2231 6th St SE
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| City |
Minneapolis |
| State/province |
MN |
| ZIP/Postal code |
55455 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE125203 |
Chromatin accessibility landscape upon induction of Msgn1, Pax3 and Myf5 in mesodermal cells and identification of conserved Pax3 binding sites and target genes during skeletal myogenesis |
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| Relations |
| BioSample |
SAMN10756592 |
| SRA |
SRX5255388 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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