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| Status |
Public on Oct 21, 2020 |
| Title |
DOT1LKO1_ESC_RNA3 |
| Sample type |
SRA |
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| Source name |
embryonic stem cell
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| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cell
cell line: v6.5
genotype/variation: DOT1L knockout/DOT1LKO
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| Treatment protocol |
EB differentiation was performed using the hanging drop method. For NPC differentiation, cells were seeded on gelatinized dish in 2i/LIF media for a day. The two inhibitors and LIF were then withdrawn from the media and cells were kept in plain N2B27 media for 7 days. Cells were then dissociated and seeded in ultra-low attachment plates (Sigma) in N2B27 media containing 10ng/ml EGF (Peprotech) and 10ng/ml FGF2 (Peprotech) for 3 days to form neurospheres. Floating aggregates (neurospheres) were then seeded on gelatinized plates in N2B27 containing 10ng/ml EGF and 10ng/ml FGF2 for 2-4 days. Attached cells were dissociated and seeded again on gelatinized plates in N2B27 containing 10ng/ml EGF and 10ng/ml FGF2. Cells were passaged one more time and harvested for different assays.
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| Growth protocol |
ESCs were grown in N2B27 media supplemented with MEK inhibitor, GSK3 inhibitor, and LIF (2i/LIF).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol reagent (Life technologies). RNAse free DNaseI (Sigma) was used to eliminate DNA contamination and the treated RNA was purified with RNeasy mini kit (Qiagen).
For ChIP, ESCs were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin were sonicated using a E220 focused-ultrasonicator (Covaris). 100 µg sheared chromatin, 5 µg antibody, and 50 µl protein A/G beads (Santa Cruz) were used for each immunoprecipitation. Immunoprecipitated DNA were purified after washing, eluting, reverse-crosslinking, and submitted for library preparation.
PRO-seq was performed following a published protocol (Mahat et al., 2016a) with minor modifications. All 4 biotinylated nucleotides were used at 25 μM each final concentration for the run-on reaction. RPPH (NEB) was used to remove the 5’ RNA cap. Libraries were size selected using a 2% agarose gel on a Pippin HT programmed to elute 140–350 bp.
ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-seq libraries were prepared using the Illumina TruSeq Stranded Total RNA Preparation Kit with Ribo-Depletion. Input RNA quality was validated using the Agilent RNA 6000 Nano Kit. 200ng-1 µg of total RNA was used as starting material. Libraries were validated using the Agilent DNA 1000 Kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
total RNA
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| Data processing |
Basecalls were performed using bcl2fastq v2.17 for NextSeq output.
Both ChIP-seq and RNA-seq reads were trimmed from 3' end until the final base had a quality score > 30, using Trimmomatic v0.33, discarding reads left with < 20 bp
ChIP-seq reads were aligned to UCSC mm9 using Bowtie version 0.12.9. Only uniquely mapping reads with at most two mismatches were retained.
RNA-seq reads were aligned to UCSC mm9 using Tophat version 2.0.9. Only uniquely mapping reads with at most two mismatches were retained.
To create ChIP-seq coverage plots, the locations of the mapped ChIP-seq reads were extended to 150 bp to represent sequenced fragments, renormalized (to reads per million, rpm) and reformatted in the bigWig file format.
To create RNA-seq coverage plots, the mapped RNA-seq reads were split according to strand, renormalized (to reads per million, rpm) and reformatted in the bigWig file format.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files were generated using the genomicRanges R package. Score represents the normalized coverage of RNA or DNA fragments at a given genomic coordinate.
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| Submission date |
Jul 10, 2019 |
| Last update date |
Oct 21, 2020 |
| Contact name |
Ali Shilatifard |
| E-mail(s) |
[email protected]
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| Organization name |
Northwestern University Feinberg School of Medicine
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| Department |
Department of Biochemistry and Molecular Genetics
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| Lab |
Shilatifard Lab
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| Street address |
320 E Superior St
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE134083 |
DOT1L-controlled cell-fate determination and transcription elongation are independent of H3K79 methylation |
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| Relations |
| BioSample |
SAMN12249577 |
| SRA |
SRX6427795 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3936468_DOT1LKO1_ESC_RNA3.minus.bw |
26.6 Mb |
(ftp)(http) |
BW |
| GSM3936468_DOT1LKO1_ESC_RNA3.plus.bw |
27.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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