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Blood, Vol. 104, Issue 1, 89-91, July 1, 2004
Deletion of the major GATA1 enhancer HS 1 does not affect eosinophil GATA1 expression and eosinophil differentiation
Blood Guyot et al.
104: 89
Supplemental materials for: Vyas, Vol 104, Issue 1, 89-91
Files in this Data Supplement:
- Table S1. Names and sequences of primers used in this study. (Adobe PDF, 59 KB) -
The approximate coordinates (in kilobases) of each PCR product are indicated relative to the transcription start site of the IE exon of the mGata1 gene. Real-time PCR primers and 5'FAM-3'TAMRA probes were designed using Primer Express 2.0 software (Applied Biosystems, Warrington, United Kingdom). Duplicate real-time PCR reactions on each immunoprecipitated template were performed on an SDS 7000 thermocycler (Applied Biosystems) using Taqman universal PCR mastermix (Applied Biosystems), 400 nM of each primer, and 200 nM of probe in 25-µL reactions. Cycling conditions were 50°C for 2 minutes and 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. All primers and probes were validated over a range of genomic DNA dilutions (1-64 ng genomic DNA) to ensure that the intensity of PCR product was proportional to the starting amount of DNA template. To calculate fold enrichment, a previously published method was used1. In histone ChiP assays, but not the transcription factor ChiP assays, this was normalized to the value obtained with GAPDH-specific primers (Eurogentec, Romsey, United Kingdom).
Reference
1. Litt MD, Simpson M, Recillas-Targa F, Prioleau MN, Felsenfeld G. Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci. Embo J. 2001;20:2224-2235
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