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| Status |
Public on Mar 21, 2020 |
| Title |
Kdm5b_KD A |
| 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 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 |
total RNA |
| Extraction protocol |
Total RNA was extracted with Trizol (Invitrogen) and rRNA depleted with Ribozero (Epicentre)
cDNA synthesis was performed by using oligo-dT as well as random hexamers. Actinomycin D was added to the reaction to prevent any possible amplification from contaminating genomic DNA. During second strand synthesis, a dU/VTP mix was used to create directional libraries. Before library preparation, cDNA samples were Covaris-fragmented to 300 bp fragments. The samples were then end-filled, 3’ terminal A-extended and ligated to pre-annealed TruSeq indexed Illumina adapters. Uracil-DNA-glycosylase (UDG) treatment preceded the PCR reaction to cleave the uridine-containing strand and therefore identify the orientation of each transcript
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
DNase-treated, rRNA-depleted mRNA
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| Data processing |
sequencing reads were aligned by the WASP pipeline version 3.1.3 (rev. 6589), using gsnap (2012-07-20)
FASTQ files incorperate Phred quality scores. NOTE: In the raw sequence data fastq file reads failing Illumina's purity filter are removed automatically
Cuffflinks was performed for each sample to calculate FPKM values, specifying the strand of the reads (fr-fisrtstrand), performing an initial estimation of the number of reads mapping in multiple locations in the genome (-u), and normalizing by the upper quartile of the number of fragments mapping to individual loci (-N). To calculate differences in expression between samples, the cufflinks results were merged by cuffmerge using a mm9 GTF file obtained from the UCSC Genome Browser as a reference genome, and then used by cuffdiff to calculate differentially expressed genes
Genome_build: mm9
Supplementary_files_format_and_content: Xlsx file containing all RefSeq genes, analyzed by Cufflinks (Cuffdiff) in order to calculate FKPM values.
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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) |
| GSE63285 |
Kdm5b depletion in embryonic stem cells does not induce intragenic promoters or affect transcription [RNA-Seq] |
| GSE63287 |
Kdm5b depletion in embryonic stem cells does not induce intragenic promoters or affect transcription |
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| Relations |
| BioSample |
SAMN03198895 |
| SRA |
SRX761370 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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