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| Status |
Public on Sep 06, 2019 |
| Title |
ATAC-seq_DAY3_2 |
| Sample type |
SRA |
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| Source name |
DAY3
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 x 129
genotype: OKSM Col1a1/M2rtTA ROSA26
Stage: DAY3
medium: ESC medium with 1ug/ml doxycycline and 50ug/ml ascorbic acid
marker: unsorted
batch: EC-DG-4663
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| Growth protocol |
Mouse ES V6.5 were cultured on irradiated feeder cells in KO-DMEM media (Invitrogen) supplemented with 15% heat-inactivated fetal bovine serum, GlutaMAX, penicillin-streptomycin, non-essential amino acids, β-mercaptoethanol and 1000 U/ml LIF (ESC medium). All V6.5 samples for HiCHIP and two replicates of ChIPseq were cultured in the presence of 2i (1uM MEKinhibitor (Stemgent 04-0006) and 3uM GSK3 inhibitor (Stemgent 04-0004)). Mouse embryonic fibroblasts (MEFs) were isolated from a "reprogrammable" mouse harboring a polycystronic OKSM cassette in the Col1a1 locus and M2rtTA in the Rosa26 locus. Cells were reprogrammed in the presence of 1ug/ml doxycycline and 50ug/ml ascorbic acid and cultured in ES medium as described above.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
50,000 cells were washed once with 50 μL of cold PBS and resuspended in 50 μL lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% (v/v) IGEPAL CA-630). The suspension of nuclei was then centrifuged for 10 min at 800 g at 4°C, followed by the addition of 50 μL transposition reaction mix (25 μL TD buffer, 2.5 μL Tn5 transposase and 22.5 μL nuclease-free H2O) using reagents from the Nextera DNA library Preparation Kit (Illumina #FC-121-103). Samples were then incubated at 37°C for 30min. DNA was isolated using a ZYMO Kit (#D4014).
ATAC-seq libraries were first subjected to 5 cycles of pre-amplification. To determine the suitable number of cycles required for the second round of PCR the library was assessed by quantitative PCR as described in Buenrostro et al and the library was then PCR amplified for the appropriate number of cycles using Nextera primers. Samples were subject to a dual size selection (0.55x-1.5x) using SPRI beads (Beckman Coulter #B23317). Finally, the ATAC libraries were sequenced on a HiSeq 2500 platform on PE50 mode.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Paired-end reads were aligned to mm10 (bowtie2 version 2.3.2, --no-unal --local --very-sensitive-local --no-discordant --no-mixed --contain --overlap --dovetail -I 10 -X 2000), and mitochondrial DNA alignments were excluded.
Reads marked as positional duplicates or falling into mouseENCODE blacklisted genomic regions (liftOver to mm10; Consortium, 2012) were filtered out.
Coverage tracks in FPKM were generated from alignments using bamCoverage (V2.3.6).
Read ends were adjusted for Tn5 transposase offsets. Peaks were called at p<10-5 (MACS version 2.1.1) per replicate, and only common peaks between two independent biological replicates were retained for further analysis.
Genome_build: mm10
Supplementary_files_format_and_content: BigWig files for coverage tracks; BED files for peak coordinates.
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| Submission date |
Apr 20, 2018 |
| Last update date |
Oct 10, 2023 |
| Contact name |
Effie Apostolou |
| E-mail(s) |
efa2001@med.cornell.edu
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| Organization name |
Weill Cornell Medicine
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| Street address |
1161 York Avenue, Apt 8A
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE113428 |
KLF4 binding is involved in the organization and regulation of 3D enhancer networks during acquisition and maintenance of pluripotency [ATAC-seq] |
| GSE113431 |
KLF4 binding is involved in the organization and regulation of 3D enhancer networks during acquisition and maintenance of pluripotency |
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| Relations |
| BioSample |
SAMN08963177 |
| SRA |
SRX3979880 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3106252_DAY3_2.bed.gz |
994.9 Kb |
(ftp)(http) |
BED |
| GSM3106252_DAY3_2.bw |
354.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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