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| Status |
Public on Nov 09, 2016 |
| Title |
ATAC_N_4DPT |
| Sample type |
SRA |
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| Source name |
Fetal Lung Fibroblast
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MRC-5 (ATCC, CCL-171)
transduced with (factor): NEUROG2
supplemented with (medium and small molecules): Neuron induction medium
atac-seq performed (#) days post treatment with small molecules: 4 DPT
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| Treatment protocol |
MRC-5 fibroblasts were seeded at a density of 0.8×10^6 cells in a 10 cm polystyrene dish. Fibroblasts were transduced with HA-NEUROG2-encoding lentivirus 24 hours after plating. The optimal lentivirus titer was empirically determined (4-fold dilution for 10 ml-capacity 10 cm dishes) and consistently yielded ≥90% TUBB3-positive and MAP2-positive neurons. Transduced-fibroblast cultures were treated with fresh medium 24 hours post infection. Cultures were transitioned to neuron induction medium (DMEM, Ham’s F12 nutrient mixture (GE Healthcare, SH30026.02), neurobasal medium (Life Technologies, 21103-049), N-2 supplement (Life Technologies, 17502‑048), B-27 supplement (Life Technologies, 17504-044), and penicillin‑streptomycin at 1 : 1 : 0.5 : 0.02 : 0.01 : 0.025).
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| Growth protocol |
MRC-5 fibroblasts were cultured in Dulbecco’s modified eagle medium (DMEM, GE Healthcare, SH30243.01) supplemented with 15% (v/v) fetal bovine serum (Corning, 35-010-CV) and 1% (v/v) penicillin-streptomycin (GE Healthcare, SV30010). Fibroblasts were maintained at 37°C in a humidified incubator with 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
MRC-5 fibroblasts were cultured, transduced with GFP-encoding, NEUROG2-encoding or NEUROG2+SOX4-shRNA-encoding lentivirus, and treated with DMEM containing 15% fetal bovine serum, neuron induction medium or neuron induction medium supplemented with 10 μM forskolin and 1 μM dorsomorphin for 4 days, respectively. Approximately 5×106 cells were treated with Accutase cell detachment solution (Innovative Cell Technologies) for 3 minutes and collected in ice-cold PBS by centrifugation (525 × g, 5 minutes, 4°C). Cells were resuspended by repeated gentle pipetting in 2 ml ice-cold resuspension buffer (PBS containing 25 mM HEPES (pH 7.0) and 5 mM EDTA). GFP expressing viable cells were isolated using fluorescence-based sorting on a MoFlo platform (Beckman Coulter). Exactly 50,000 cells were sorted into lysis buffer (10 mM Tris (pH 7.5), 10 mM NaCl, 3 mM MgCl2, and 0.1% (v/v) IGEPAL CA 630) at 4°C then collected by centrifugation (21,130 × g, 2 minutes, 4°C). The pellet was resuspended in 50 μl transposition mix (25 μl 2X TD buffer, 22.5 μl nuclease-free H2O, and 2.5 μl Nextera Tn5 transposase (Illumina, FC-121-1030)) and incubated exactly 30 minutes at 37°C with gentle agitation (400 rpm). Chromatin was purified with a MinElute PCR purification kit (Qiagen, 28004) and twice eluted in 10 μl elution buffer.
Amplification reactions of 20 μl eluted chromatin, 2.5 μl universal ATAC primer (25 μM, Appendix 4), 2.5 μl barcoded ATAC primer (25 μM, Appendix 4), and 25 μl NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs, M0541S) were amplified for 1 cycle (5 minutes, 72°C) followed by 5 cycles (10 seconds, 98°C; 30 seconds, 63°C; 1 minute, 72°C). To determine the number of additional amplification cycles required, quantitative real-time PCR using 5 μl ATAC library reaction, 2.5 μl NEBNext High-Fidelity 2X PCR Master Mix, 0.125 μl universal ATAC primer (25 μM), 0.125 μl barcoded ATAC primer (25 μM), and 2 μl 5X SYBR Green I (ThermoFisher Scientific, S7563) was performed for 1 cycle (30 seconds, 98°C) followed by 20 cycles (10 seconds, 98°C; 30 seconds, 63°C; 1 minute, 72°C). The appropriate number of additional amplification cycles was determined for each library (11 total cycles) and amplified libraries were purified with a MinElute PCR purification kit then eluted in 20 μl elution buffer. Right side size selection with 0.4X Agencourt AMPure XP beads (Beckman Coulter, A63881) was used to reduce fragments larger than 1,000 nucleotides and left side size selection with 1X Agencourt AMPure XP beads was used to eliminate fragments smaller than 150 nucleotides. Fragment length and size selection were evaluated after library amplification using a high-sensitivity DNA analysis kit (Agilent Technologies, 5067-4626). Library quantification prior to flow cell loading was performed using bioanalyzer traces and a Quant-iT PicoGreen dsDNA assay kit (ThermoFisher Scientific, P11496).
ATAC-seq: single end 50-base length sequencing reads were generated on an Illumina HiSeq 2500 System.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Genome_build: GRCh37/hg19
Reads were aligned to the human reference sequence GRCh37/hg19 with the Bowtie algorithm (version 1.0.0). Peak calling, peak‑gene annotation, motif discovery (parameter: 200 nucleotide window from peak center), and generation of heatmap matrices were performed using HOMER (version 4.7). The UCSC Genome Browser was used to visualize tag densities and multi-experiment datasets.
Supplementary_files_format_and_content: Alignment files (.bam) were generated using Bowtie (version 1.0.0). UCSC.bedGraph and peak files (.txt) were generated using HOMER (version 4.7)
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| Submission date |
Apr 25, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Derek K Smith |
| E-mail(s) |
smith.5822@osu.edu
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| Organization name |
The University of Texas Southwestern Medical Center
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| Department |
Molecular Biology
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| Lab |
Chun-Li Zhang, Ph.D.
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| Street address |
5323 Harry Hines Blvd, NA5.202
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| City |
Dallas |
| State/province |
Texas |
| ZIP/Postal code |
75390 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE75912 |
Small molecules modulate chromatin accessibility to promote NEUROG2-mediated fibroblast-to-neuron reprogramming |
| GSE80639 |
Small molecules modulate chromatin accessibility to promote NEUROG2-mediated fibroblast-to-neuron reprogramming [ATAC-seq] |
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| Relations |
| BioSample |
SAMN04893752 |
| SRA |
SRX1725545 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2132060_ATAC_N_4DPT_peaks.txt.txt.gz |
1.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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