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| Status |
Public on Jun 05, 2012 |
| Title |
MCF7 siCONTROL |
| Sample type |
SRA |
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| Source name |
Human breast adenocarcinoma cell-line MCF7
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
passage: 7
sirna: ON-TARGETplus Non-Targeting siRNA Control (Dharmacon, D-001810-01)
treatment: control
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| Treatment protocol |
1. Low passage MCF7 cells were grown in DMEM supplemented with 10% FBS (no Pen/Step) to 85% confluency and passaged into 6-well dish at approximately 50% confluency 2. Cells were transfected with final concentration 50nM siRNA using Lipofectamine2000 according to the manufacturer's instructions (Invitrogen) 3. The media was replaced 12 hours post transfection and cells were grown for an additional 36 hours (hours posttransfection)
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| Growth protocol |
Thawing Cells: 1. Thaw vial immediately in 37º C water bath. Keep O ring above the water surface to prevent contamination. Thaw content with slight shake until only small ice is left in vial. It usually takes 1 min. Spray vial with 70% ethanol all over and wipe its surface with clean tissue in the hood. 2. Open the vial and transfer the content to a 15 ml Falcon tube already containing 5 ml of fresh medium. 3. Spin down at 1000 rpm or 200g for 3 to 5 mins at 4º C. Aspirate supernatant. 4. Resuspend cells in fresh medium and transfer to 150mm x 25mm tissue culture dish. 5. Check the cells under microscope. 6. Cells are cultured in CO2 incubator and medium is changed about every 3 days. 7. It usually takes 3 days or more for cells to recovery from freezing. After cell culture reaches 80-85% confluence, subculture is conducted. Subculture ratio is about 1:3 or 1:4. Passaging Cells: 1. Observe cells to see how confluent they are, whether the cells are alive, whether the cells are contaminated, and whether the cells have the correct morphology. After cell culture reaches 80 to 85% confluence, subculture is conducted 2. Remove media from dish 3. Wash 1x with 10 ml of PBS 4. Add 5 ml of Trypsin and trypsinize for 3 min at 37C. Whack hard – you should see the cells coming down. (Important: never overtrypsinize the cells, so work quickly) 5. Add 5 ml of media and use it to rinse the dish to detach the cells off (4 to 5 times) 6. Spin down at 1000rpm for 3 to 5 min at room temperature. Aspirate supernatant 7. Add 15 ml of media to 15ml tube containing cell pellet, and pipette up and down to mix 8. Add 15 ml of media to each new 150mm x 25mm tissue culture dish (we split 1 dish into 3 to 4 dishes) 9. Add 5mls of cell culturecontaining media to each tissue culture dish. Make sure the media covers the entire area of the dish. Put the cells into 37C with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Trizol Reagent (Life Technologies) following the suggested protocol; 2ug of each RNA sample was used with the Illumina TruSeq RNA Sample Prep Kit (Cat # RS-122-2001) to make RNA libraries following the Illumina TruSeq RNA Sample preparation Low-Throughput protocol. Briefly, RNA was fragmented, then first-strand cDNA was prepared using the kit supplied 1st Strand Master Mix and user-supplied Superscript III (Life Technologies, cat # 18080-051) followed by second strand cDNA synthesis. The Illumina protocol and reagents were used to complete the library preparation, with 12 cycles of PCR amplification.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg19 whole genome using tophat with default parameters
Fragments Per Kilobase of exon per Megabase of library size (FPKM) were calculated using a protocol as described in Trapnell et al, Nature Protocols 7, 562–578 (2012). In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample
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| Submission date |
Jun 04, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
rui wang |
| E-mail(s) |
ruiw2009@gmail.com
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| Phone |
614-292-6931
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| Fax |
614-688-6600
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| Organization name |
Ohio State University
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| Street address |
212 Biomedical Research Tower,460 West 12th Ave
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| City |
Columbus |
| State/province |
OHIO |
| ZIP/Postal code |
43210 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE38447 |
Cell type-specific binding patterns reveal that TCF7L2 can be tethered to the genome by association with GATA3 |
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| Relations |
| SRA |
SRX151408 |
| BioSample |
SAMN01036671 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM942209_USC229_genes_fpkm_tracking.txt.gz |
660.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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