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| Status |
Public on Mar 21, 2020 |
| Title |
Kdm5b_KD_B |
| Sample type |
SRA |
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| Source name |
WW6 cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: WW6
cell type: embryonic stem cells
strain: ~20% C57/B6J, ~75% 129/Sv and ~5% SJL
treatment: transfected cells with Kdm5b targeting construct
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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 |
mES cells were collected by washing and trypsinizing for three minutes, and FACS sorted by the GFP signal present in the transfected plasmid. Genomic DNA was extracted by resuspending the cells in lysis buffer (10 mM TE, 150 mM EDTA and 1% SDS) supplemented with 10 mg/ml of RNase A and 20 mg/ml of Proteinase K, and incubated at 50 °C overnight. The lysed cells were phenol-chloroform extracted and the resultant DNA was dialyzed in 0.2X SSC buffer (300 mM NaCl and 3 mM Na3C6H5O7, ph 7.0) for 24 hours. The DNA sample was concentrated in the dialysis bags using polyethylene glycol, following which the quality and concentration of the DNA were measured by Nanodrop spectrophotometry.
Two custom adapters were created for HELP-tagging, referred to as AE and AS. As well as an Illumina adapter sequence, adapter AE contains an EcoP15I recognition site and a T7 promoter sequence. Adapter AS contains an Illumina sequencing primer sequence. One micrograms of genomic DNA were digested with HpaII and MspI in separate 50 μl reactions and purified by phenol/chloroform extraction followed by ethanol precipitation. The digested genomic DNA was ligated to adapter AE using an NEB Quick Ligation Kit (25 μl of 2x Quick ligase buffer, 1 μg of MspI/HpaII-digested DNA , 0.1 μl of Adapter AE (1 μM), 3 μl of Quick Ligase in a final volume of 50 μl). After AE ligation, the products were purified using AMpure (Agencort), then digested with EcoP15I (NEB). The restriction fragments were end-repaired to inhibit dimerization of adapters, and tailed with a single dA, at the 3’ end. After the dA tailing reaction, adapter AS was ligated to the dA-tailed fragments using an NEB Quick Ligation Kit (25 μl of 2x Quick ligase buffer, 2.5 μl of Adapter AS (10 μM), 2.5 μl of Quick Ligase in a final volume 50 μl). After ligation, products were purified using the MinElute PCR purification kit (Qiagen) and in vitro-transcribed using MEGAshortscript (Ambion). Following in vitro transcription, products were purified with an RNeasy clean-up kit (Qiagen) before reverse transcription was performed using the SuperScript III kit (Invitrogen). The first strand cDNA produced was used as a template for PCR using the following conditions: 98°C for 2 minutes, then 18 cycles of 98°C for 15 seconds, 60°C for 15 seconds and 72°C for 15 seconds followed by 5 minutes at 72°C for the final extension. The product then underwent gel extraction (Qiagen).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
library strategy: HELP-tagging
Images generated by the Illumina sequencer were analyzed by Illumina pipeline software (versions ELAND - 1.7.0). Initial data processing was performed using the default read length of 36 bp. We permitted up to two mismatches in each sequence, and allowed a sequence to align to up to a maximum of 10 locations within the genome. For non-unique alignments, a sequence was assigned a partial count for each aligment location amounting to 1/n, where n represents the total number of aligned positions. To normalize the data between experiments, the number of sequences associated with each HpaII site was divided by the total number of sequences (including partial counts) aligning to all HpaII sites in the same sample.
Genome_build: mm9
Supplementary_files_format_and_content: The hit count files were generated using in house pipeline (WASP - 3.1.4); The first line starts with "total" and displays total number of raw reads aligned to HpaII loci properly. For instance, "total 5249033" ; From the second line onward, each line represents one HpaII locus, showing the ID, chromosome, offset, HpaII count(F strand), HpaII count(R strand), HpaII count(both strands), respectively and space-delimited. For instance, "21 chr1 11961 9.8 17 26.8"
Supplementary_files_format_and_content: A bedgraph of the HpaII loci angle values was generated for each sample using custom script provided in the publication; Angle values represent degree of unmethylation at each HpaII site
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| Submission date |
Nov 18, 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) |
| GSE63287 |
Kdm5b depletion in embryonic stem cells does not induce intragenic promoters or affect transcription |
| GSE63434 |
Kdm5b depletion in embryonic stem cells does not induce intragenic promoters or affect transcription [HELP-tagging] |
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| Relations |
| BioSample |
SAMN03199799 |
| SRA |
SRX763255 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1548731_HT_HpaII_siKdm5b_B.mm9.angle.bedgraph.gz |
6.3 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM1548731_HT_HpaII_siKdm5b_B.mm9.hcount.txt.gz |
4.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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