| Sample GSM801643 | Query DataSets for GSM801643 |
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| Status | Public on Jun 01, 2012 |
| Title | GATA1 KD CMK-5a-1 |
| Sample type | RNA |
| Channel 1 |
| Source name | CMK neg control cells |
| Organism | Homo sapiens |
| Characteristics | transfection: negative controlclone: CMK-negparental cell line: CMK |
| Treatment protocol | Knockdown of GATA1 in the CMK cells was performed using shRNA lentivirus. Two clones designated CMK-5a and CMK-5b were used. A pool of cells from a negative control infection (lentivirus expressing a shRNA with limited homology to any known human genes) was used as the negative control (designated CMK-neg). |
| Growth protocol | The parental CMK cells and the shRNA stable clones were cultured in RPMI 1640 with 10% fetal bovine serum (Hyclone, Logan, UT) and 2 mM L-glutamine plus 100 U/ml penicillin and 100 µg/ml streptomycin, in a 37 °C humidified atmosphere containing 5% CO2/95% air. |
| Extracted molecule | total RNA |
| Extraction protocol | Total RNAs were extracted from the CMK shRNA stable clones using TRIzol reagent (Invitrogen, Carlsbad, CA). The quality and quantify of the extracted total RNAs were determined on a NanoDrop analyzer. |
| Label | Alexa 555 |
| Label protocol | Alexa555 and Alexa 647 labeled aRNA was prepared from 500ng total RNA using the Epicentre TargetAMP 1-Round Aminoallyl-aRNA Amplification Kit 101 according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Alexa Dye (Invitrogen/Molecular Probes, Eugen OR) incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer. |
| Channel 2 |
| Source name | CMK-5a GATA1 shRNA infected cells |
| Organism | Homo sapiens |
| Characteristics | transfection: GATA1 shRNAclone: CMK-5Aparental cell line: CMK |
| Treatment protocol | Knockdown of GATA1 in the CMK cells was performed using shRNA lentivirus. Two clones designated CMK-5a and CMK-5b were used. A pool of cells from a negative control infection (lentivirus expressing a shRNA with limited homology to any known human genes) was used as the negative control (designated CMK-neg). |
| Growth protocol | The parental CMK cells and the shRNA stable clones were cultured in RPMI 1640 with 10% fetal bovine serum (Hyclone, Logan, UT) and 2 mM L-glutamine plus 100 U/ml penicillin and 100 µg/ml streptomycin, in a 37 °C humidified atmosphere containing 5% CO2/95% air. |
| Extracted molecule | total RNA |
| Extraction protocol | Total RNAs were extracted from the CMK shRNA stable clones using TRIzol reagent (Invitrogen, Carlsbad, CA). The quality and quantify of the extracted total RNAs were determined on a NanoDrop analyzer. |
| Label | Alexa 647 |
| Label protocol | Alexa555 and Alexa 647 labeled aRNA was prepared from 500ng total RNA using the Epicentre TargetAMP 1-Round Aminoallyl-aRNA Amplification Kit 101 according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Alexa Dye (Invitrogen/Molecular Probes, Eugen OR) incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer. |
| Hybridization protocol | Whole human genome oligonucleotide microarrays (Agilent Technologies, catalog no. G4112A) were hybridized using 0.75 ug of Alexa 555 labeled aRNA and 0.75 ug of Alexa 647 labeled aRNA. Agilent's SureHyb hybridization chambers were used in a hybridization oven and rotation rack for 17 h at 60 °C. After hybridization, the slides were washed per Agilent's SSPE wash protocol. |
| Scan protocol | Slides were scanned using an Agilent dual laser scanner with extended dynamic range (XDR) at 5 micron resolution. |
| Data processing | Tiff images were analyzed with Agilent's feature extraction software (v9.5.3.1) using protocol GE2_v5_95_Feb07. Linear and LOWESS normalization were performed. |
| Submission date | Sep 26, 2011 |
| Last update date | Jun 01, 2012 |
| Contact name | Yubin Ge |
| E-mail(s) | [email protected] |
| Phone | 313 578-4285 |
| Organization name | Karmanos Cancer Institute |
| Street address | 110 East Warren Ave. |
| City | Detroit |
| State/province | MI |
| ZIP/Postal code | 48201 |
| Country | USA |
| Platform ID | GPL4133 |
| Series (1) | | GSE32388 | The unique role of GATA1s in Down syndrome acute megakaryocytic leukemia biology and therapy |
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