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| Status |
Public on May 15, 2020 |
| Title |
ATAC_D8_iTSC_rep_1 |
| Sample type |
SRA |
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| Source name |
human fibroblasts
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| Organism |
Homo sapiens |
| Characteristics |
cell type: iTSCs
passages: P28
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| Growth protocol |
To generate iTSCs used for this study, somatic cell reprogramming with the Cytotune 2.0 kit, experiments were performed according to the manufacturer’s instructions (Invitrogen). Briefly, human dermal fibroblast (adult) were seeded at ~5x104 cells per well of a 6-well plate in fibroblast medium containing DMEM (Gibco), 10% FBS (Hyclone), 1% nonessential amino acids (Gibco), 1mM GlutaMAX (Gibco), Pen-strep (Gibco), 0.1mM 2-mercaptoethanol (Gibco) and 1mM sodium pyruvate (Gibco) . After 2 days, cells were transduced with Sendai viruses in fibroblast medium with multiplicity of infections (MOIs) as follows, KOS MOI=5, c-MYC MOI=15, KLF4 MOI=6. After 24 hours, the medium was replaced with fresh fibroblast medium and media were changed every other day thereafter. On day 7, cells were trypsinized and seeded onto a layer of iMEF feeders at ~0.5x105~1x105 per flask in fibroblast medium. The next day, the cells were switched either to TSC media for d8 iTSCs, or to naive media for d21N iTSCs, 21-28 days after transduction, cells were passaged and expanded.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
iTSCs were isolated by FACS (see Methods) and ~65k cells were washed and lysed in ATAC-seq lysis buffer (10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630, 10 mM Tris pH 7.4). The transposition reaction was then carried out by using 22.5 μl of UltraPure Distilled Water (ThermoFisher, Cat#10977-015), 25 μl of Tagment DNA Buffer (Illumina, Cat#15027866) and 2.5 μl of Tagment DNA Enzyme 1 (Illumina, Cat#15027865) for each sample, and then incubated for 30 min at 37°C, followed by immediate purification using a MinElute Reaction Cleanup Kit (Qiagen, Cat#28204) according to the manufacturer’s instructions.
11 μl of transposed DNA, 25μl of the NEBNext High-Fidelity 2x PCR Master Mix (Cat#M0541S) and 1.25μM of the adaptor sequences as published previously were used in a 50 μl PCR reaction. PCR parameters were: 72°C for 5 min, 98°C for 30 s and 9 cycles of 98°C for 10 s, 63°C for 30 s and 72°C for 1 min. The prepared libraries were purified using a MinElute PCR purification kit (Qiagen, Cat#28004) followed by Agencourt AMPure XP beads (Beckman Coulter, Cat#A63880) according to the manufacturer’s specifications, where library fragments ranging from 200 to 700 bp were selected and sequenced on an Illumina HiSeq 1500 in 2x51 cycle paired-end mode
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Data processing |
ATAC sequencing reads (pair-end 51nt reads) were adaptor-trimmed and filtered by base quality and length using Cutadapt v 1.852 using -a CTGTCTCTTATACACATCT, -A CTGTCTCTTATACACATCT, -q 20, and --minimum-length 18 options. Read pairs passing filters were mapped to the complete human genome [hg19 human genome (UCSC version, December 2011)] using Bowtie2 with -X 2000, --no-mixed and --no-discordant options.
Mapped sample reads were filtered for multi-mappers (mapping quality < 10) and reads mapped to mitochondrial DNA using Jvarkit’s samjs. PCR duplicates were discarded using Picard’s (http://broadinstitute.github.io/picard) MarkDuplicates tool. Sequencing reads aligned to known genomic blacklisted regions were also not considered for further analysis
Peak calling was performed on each biological replicate with MACS2 callpeak subcommand using --nomodel -f BAM --keep-dup all --gsize hs --shift -100 --extsize 200 --SPMR -B options. For downstream analysis we use an “intersect and rescue” approach. This approach consisted of intersecting each time point and reprogramming media biological replicates peak sets (bedtools intersect), subcommand (-wa -wb -F/-f 0.3) and then filtering those peaks with a fold change over background of more than 5 fold change (FC) and at least 3 FC in the other replicate. This created two intersection peaksets (major to 5 FC in replicate 1 and major to 3 FC in replicate 2 and vice versa), which were then combined and merged with bedtools merge (a minimum of 1 bp overlap). The union peakset of both replicates for each timepoint and reprogramming media was then reduced by merging all peaks within 100 bp. Finally, a consensus peak set of all time points and reprogramming media was created using bedtools merge as described above.
Genome_build: hg19
Supplementary_files_format_and_content: narrowPeak files are the output of macs2 callpeaks. Format is is BED6+4 (details about the format can be found at https://genome.ucsc.edu/FAQ/FAQformat.html#format12 and https://github.com/taoliu/MACS/). Macs2 specific columns are: 5th display integer score (int(-10*log10qvalue)), 7th fold change at peak summit relative to background, 8th -log10pvalue at peak summit, 9th -log10qvalue at peak summit and 10th relative peak summit position to peak start. Detailed information can be found at MACS webiste (https://github.com/taoliu/MACS/). BigWig files were created using UCSCs bedGraphToBigWig from the normalised bedgraph file output from MACS2.
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| Submission date |
May 14, 2020 |
| Last update date |
May 15, 2020 |
| Contact name |
Fernando Rossello |
| E-mail(s) |
[email protected], [email protected]
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| Phone |
+61 3 85598749
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| Organization name |
The University of Melbourne
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| Street address |
L 10, 305 Grattan Street
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| City |
Melbourne |
| State/province |
VIC |
| ZIP/Postal code |
3000 |
| Country |
Australia |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE150590 |
Chromatin accessibility analysis of induced trophoblast stem cells. |
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| Relations |
| BioSample |
SAMN14928106 |
| SRA |
SRX8343791 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4552868_32F_d8TSC_S1_peaks.narrowPeak.gz |
3.4 Mb |
(ftp)(http) |
NARROWPEAK |
| GSM4552868_32F_d8TSC_S1_treat_pileup.bw |
326.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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