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| Status |
Public on Feb 01, 2018 |
| Title |
Mll4dSET_FBS_RNA_3 |
| 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: MLL4 SET deletion/MLL4dSET
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| Treatment protocol |
For induction of the exit of naive pluripotency, 2i+LIF cultured ESCs were passaged to 0.1% gelatin (Stem cell technologies) coated plates and cultured for more than 2 passages in FBS+LIF media containing Dulbecco's modified Eagle's medium (DMEM) (Corning) supplemented with 15% FBS (Gemini), 1×Penicillin-Streptomycin (Life technologies), 1× Glutamax (Life technologies), 1× MEM non-essential amino acids (Life technologies), 1× β-mercaptoethanol (Life technologies), and 1.8×103 U/ml of LIF (Gemini).
For shRNA knockdown, ESCs were infected with lentivial shRNA in the presense of 10ug/ml polybrene for 12 hours and then selected with 2 ug/ml puromycin for 48h before harvest for ChIP-seq or RNA-seq experiments.
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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, and reverse-crosslinking and submitted for library preparation.
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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| 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 |
May 17, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Ali Shilatifard |
| E-mail(s) |
ash@northwestern.edu
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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) |
| GSE99022 |
An Mll4/COMPASS-Lsd1 epigenetic axis governs enhancer function and pluripotency transition in embryonic stem cells |
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| Relations |
| BioSample |
SAMN07138473 |
| SRA |
SRX2831970 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2630481_Mll4dSET_FBS_RNA_3.minus.bw |
70.3 Mb |
(ftp)(http) |
BW |
| GSM2630481_Mll4dSET_FBS_RNA_3.plus.bw |
73.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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