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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 11, 2019 |
| Title |
WT-BAP1 |
| Sample type |
SRA |
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| Source name |
mouse ES cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: cultured ES cells
strain: 129SVxC57/BL6
passages: passages 3-5
antibody: Santa Cruz BAP1 C-4, Lot B2117
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| Growth protocol |
Co-derived wild-type and Srap1 KO mouse ES cells were cultured on gelatin-coated plates in high-glucose DMEM containing 15% FBS, 1x non-essential amino acids, 2 mM L-glutamine, 100 mM beta-mercaptoethanol, 1x nucleosides (Millipore), 1000U/ml leukemia inhibitory factor (StemRD) and 2i (selective GSK3β & Mek 1/2 inhibitors; Millipore), 25 mg/ml L-ascorbic acid.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP, MeDIP and RNA library preparation For ChIP, strain-matched wild-type or Mettl4 KO mouse ES cells (C57BL/6N x 129/Sv background) were fixed in 1% paraformaldehyde for 10 min at 37°C, followed by quenching with 125 mM glycine for 5 min. Cells were lysed and processed using the MAGnify ChIP system (Life Sciences). Lysates were sonicated using an ultrasonic processor (Cole Parmer) at power setting 3 in 30 s pulses followed by 1 min on ice, for a total of 6 min sonication. Nucleoprotein complexes were immunoprecipitated the indicated antisera (2.5 g) equivalent amounts of isotype-matched control IgG per sample. For MeDIP experiments, genomic DNA was isolated from ES cells using the DNeasy Blood & Tissue DNA kit and sonicated for a total of 6 min. Samples were processed using the hMeDIP kits (Active Motif) according to the manufacturer’s protocol. For RNA-seq, total RNA was extracted from mouse ES cells using the Zymo RNA Clean & Concentrator (Zymo Research), followed by mRNA enrichment and cDNA preparation using the KAPA Stranded RNA-Seq Kit. Library preparations were performed using the Kapa HyperPrep kit, followed by 12 cycles of PCR amplification with Kapa HiFi HotStart polymerase. Amplified libraries were pooled, purified twice with 0.9X Ampure XP beads and analyzed on an Illumina HiSeq2000 or NextSeq500.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
For ChIP-seq, DIP-seq and RNA-seq, raw reads were trimmed to remove adapters and low- quality reads using Trim Galore (v0.3.5). Bowtie (v0.12.8) was used to obtain reads mapped uniquely to the mouse genome (mm10). ChIP and DIP peak candidates were identified with MACS (v2.1.1) using input as the control data set. To remove nonspecific signals, IgG samples were processed similarly and their normalized read density (RPKM) values were subtracted from modification-specific peaks. For DIP, the empirical FDR was estimated by exchanging the DIP and IgG control samples and identifying peaks in the control sample using the same set of parameters used for the DIP sample. Called peaks were annotated with Gencode M1 and Cistrome CEAS (Shin et al., 2009) . Normalized read density plots centered on annotated elements downloaded from ENCODE were generated using NGSPLOT (Shen et al., 2014) . Peak overlap statistics were calculated using Fisherbed. For RNA-seq, Cufflinks (v 2.2.1.0) was used to assemble transcripts, estimate their abundances and test for differential transcript expression. All GO analyses used DAVID (http://david.abcc.ncifcrf.gov).
Genome_build: mm10
Supplementary_files_format_and_content: bam files were mapped using TopHat, FPKM values calculated with CuffLinks
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| Submission date |
Oct 29, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Douglas Edmund Feldman |
| E-mail(s) |
defeldma@usc.edu
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| Organization name |
USC Keck School of Medicine
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| Department |
Pathology
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| Lab |
HMR 212
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| Street address |
2011 Zonal Avenue
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90033 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE105006 |
An Adversarial DNA N6-methyladenine-Sensor Network Preserves Polycomb Silencing |
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| Relations |
| BioSample |
SAMN07945307 |
| SRA |
SRX3341233 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2835744_SK-Chip-Seq-4_S36_L004_R1_001.bigwig |
69.7 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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