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| Status |
Public on Mar 21, 2020 |
| Title |
Control_Input |
| Sample type |
SRA |
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| Source name |
WW6 cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic stem cells
strain: ~20% C57/B6J, ~75% 129/Sv and ~5% SJL
treatment: transfected cells with mock control construct
chip antibody: none
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| Treatment protocol |
Cells were transiently transfected after 24 hours of seeding, using 6 µl of Lipofectamine LTX supplemented with 5 µl of Plus reagent per ml (Invitrogen), in serum-free media (OPTI-MEM, Invitrogen), with siRNA targeting Kdm5b, or GFP as control. After 4 hours of transfection, regular cultured media was added to transfected cells to assure optimal culture conditions.
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| Growth protocol |
mES cells were grown in a feeder cell-free environment on 0.1% gelatin covered dishes, in Dulbecco’s modified Eagle medium (DMEM, Mediatech) supplemented with 10% Fetal Bovine Serum (STEMCELL Technologies cat. no. 06952), 1X Nonessential amino acids (Gibco, cat.no. 11140-050), 1X Nucleoside mix (Sigma), 55 nM 2-Mercaptoethanol (Gibco) and 3,000 units of Leukemia inhibition factor (EMD Millipore ESG1106) per ml, at 37 °C in 5% CO2
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
3 million cells crosslinked in 1% formaldehyde were lysed with Farnham and RIPA cold lysis buffers. Following collection, the crude nuclear preparation was processed in a Bioruptor at the high setting for a total of 30 minutes, in cycles of 30 seconds on/30 seconds off. The chromatin was collected by centrifugation and incubated with 5 µg of H3K4me3 antibody overnight.
The immunoprecipitated samples were end-filled using a combination of T4 DNA polymerase and T4 polynucleotide kinase , 3’ -terminal-A extended with Klenow 5'-3' exo minus, and ligated to pre-annealed TruSeq indexed Illumina adapters. Libraries were amplified using Illumina primers and gel extracted for size selection and primer-dimer removal. Before sequencing, libraries were tested using the BioAnalyzer to test quality in terms of size and primer-dimer depletion.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Poor quality bases and adapter sequences were trimmed from reads using Trim Galore! v. 0.3.3 - default parameters.
The trimmed reads were then aligned using Burrows-Wheeler aligner (BWA) v. 0.7.9a to the mm9 mouse genome. First the aln command was used to create a .bai file. Then the samse command using the .bai and original fastq files as input resulted in a .sam file containing the aligned reads
Output SAM files were converted to .bam files and sorted using Picard tools v. 1.117 using the SortSam command with the following parameters: SO=coordinate TMP_DIR=. CREATE_INDEX=true VALIDATION_STRINGENCY=LENIENT
Peaks were called using MACS2 v. 2.1.0.20140616 (callpeak command and -g mm parameter) and the proper .bam files for treatment and control (input). Peaks are in .bed format
Genome_build: mm9
Supplementary_files_format_and_content: bed
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| Submission date |
Nov 14, 2014 |
| Last update date |
Mar 21, 2020 |
| Contact name |
Andrew Johnston |
| E-mail(s) |
Andrew.Johnston@med.einstein.yu.edu
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| Organization name |
Albert Einstein College of Medicine
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| Department |
Genetics
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| Lab |
Price 314
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| Street address |
1301 Morris Park Avenue
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| City |
Bronx |
| State/province |
NY |
| ZIP/Postal code |
10461 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE63286 |
Kdm5b depletion in embryonic stem cells does not induce intragenic promoters or affect transcription [ChIP-Seq] |
| GSE63287 |
Kdm5b depletion in embryonic stem cells does not induce intragenic promoters or affect transcription |
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| Relations |
| BioSample |
SAMN03198891 |
| SRA |
SRX761364 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data not applicable for this record |
| Raw data are available in SRA |
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