title: Illumina HiSeq 2000 sequencing; Polycystic Kidney Disease Progression Expression Analysis
design: Polycystic Kidney Disease Progression Expression Analysis
library_name: cKO_Pkd1cko-11wk_2_s
library_construction_protocol: RNA sequencing was done on kidneys from 5 Wild-type (Wt) mice and 24 iKsp-Pkd1del mice with gene disruption at the age of 38-90 days (Mutant). 4 mice per group were sacrificed at 2wk, 3wk and 6wks of age. 5 mice were sacrificed  at 11wk of age, 4 at 12wk of age and 3 mice at 15wk of age.  Pkd1-cKO mice have a homozygously bred kidney–specific, tamoxifen–inducible Cre and two LoxP sites that flank exons 2–11 of the Pkd1 gene. These mice were all on full C57BL6/J genetic background. he mice received 200 mg/kg tamoxifen at P40–P42, 150 mg/kg tamoxifen at P18–P20, or 6 mg/kg tamoxifen at P10–P12. Tamoxifen administration at these dosages and ages is well tolerated on the basis of careful assessment of behavior, but the P10–P12 mice have reduced body weights caused by the tamoxifen treatments.  Male iKsp-Pkd1del mice were initially fed regular rodent chow. Approximately 7 days after tamoxifen administration, animals in the treatment groups received sirolimus-supplemented chow (Wyeth Pharma) in a dose of 100 mg/kg chow (high dose) or 10 mg/kg chow (low dose). Sirolimus was mixed with regular chow (SSNIFF Spezialdiaeten). Control mice were fed the same chow without supplementation. Mice were treated for 79 days (approximately 80 days) or 105 days. In addition, sirolimus treatment was started in two groups at 87 days upon Pkd1 gene disruption and the animals were treated with either high- or low-dose sirolimus for 105–110 days Curcumin (∼70% purity) was obtained from Sigma (Zwijndrecht, The Netherlands) and RM3(P) pellets with 1% curcumin or without curcumin were prepared by Special Diets Services (Witham, UK). This dose and way of administration were chosen in analogy to a recent experimental study that studied the effectiveness of curcumin to inhibit prostate carcinoma [PMID: 18437538 ] Mice were treated twice a week with ip injections of 1, 3, or 10 mg/kg sActRIIB-Fc in PBS. As a control group, mice received ip injections of PBS twice a week. mRNA was isolated from total RNA using the oligodT magnetic beads. After fragmentation of the mRNA, a cDNA synthesis was performed. Technical replicates followed exactly the same procedure of sample collection, dna extraction and library construction and were run on a different flow cell in the same method mRNA was isolated from total RNA using the oligodT magnetic beads. After fragmentation of the mRNA, a cDNA synthesis was performed. This was used for ligation with the sequencing adapters and PCR amplification of the resulting product. The quality and yield after sample preparation were measured with a DNA 1000 Lab-on-a-Chip (Agilent Technologies). The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp on a DNA 1000 chip).
attributes.tag: Experimental Factor: disease
attributes.value: Autosomal dominant polycystic kidney disease
attributes.tag: Experimental Factor: compound
attributes.value: tamoxifen
attributes.tag: Experimental Factor: dose
attributes.value: 200
attributes.tag: Experimental Factor: stimulus
attributes.value: none