title: PG283 array

description: PG283 is an illegitimate diploid from a mating between an rad52 mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a rad52 mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from PG283 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in PG283.

source_name: PG283 chromosomal DNA labeled with Cy5


title: PG271 array

status: Public on Jun 18 2009

type: genomic

city: Durham

first: Anne

last: Casper

state: NC

address: 381 CARL Building, Research Drive, Box 3054

department: Department of Molecular Genetics and Microbiology

country: USA

institute: Duke University

description: PG271 is an illegitimate diploid from a mating between an rad52 mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a rad52 mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from PG271 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in PG271.

hyb_protocol: Described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005).

scan_protocol: Array scanned in Genepix 4000b scanner.

data_processing: The log2 R/G ratios for all probes in the array were collapsed to a single value in the case of repeated probes and normalized according to the median value for the entire array.

molecule: genomic DNA

organism: Saccharomyces cerevisiae

source_name: PG271 chromosomal DNA labeled with Cy5

label_protocol: Direct incorporation with klenow polymerase and random priming

extract_protocol: Genomic DNA was harvested by standard protocol:  Cells were treated with zymolyase to obtain spheroblasts.  SDS was used to disrupt the cell membrane.  Potassium acetate was used to precipitate protein and lipids.  DNA was precipitated with isopropanol.  RNAse A was used to degrade RNA.

tag: strain

tag: ploidy

value: diploid

tag: recombination

value: illegitimate

source_name: MS71 chromosomal DNA labeled with Cy3

tag: isogenicity

value: isogenic to the strain in Channel 1


title: DAMC560 array

description: DAMC560 is an illegitimate diploid from a mating between an mre11::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a mre11::HPH mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC560 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC560.

source_name: DAMC560 chromosomal DNA labeled with Cy5


title: DAMC561 array

description: DAMC561 is an illegitimate diploid from a mating between an mre11::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a mre11::HPH mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC561 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC561.

source_name: DAMC561 chromosomal DNA labeled with Cy5


title: DAMC555 array

description: DAMC555 is an illegitimate diploid from a mating between an mre11::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a mre11::HPH mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC555 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC555.

source_name: DAMC555 chromosomal DNA labeled with Cy5


title: DAMC553 array

description: DAMC553 is an illegitimate diploid from a mating between an mre11::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a mre11::HPH mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC553 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC553.

source_name: DAMC553 chromosomal DNA labeled with Cy5


title: PG270 array

description: PG270 is an illegitimate diploid from a mating between an rad52 mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a rad52 mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from PG270 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in PG270.

source_name: PG270 chromosomal DNA labeled with Cy5


title: DAMC550 array

description: DAMC550 is an illegitimate diploid from a mating between an sae2::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a sae2::HPH mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC550 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC550.

source_name: DAMC550 chromosomal DNA labeled with Cy5


title: DAMC549 array

description: DAMC549 is an illegitimate diploid from a mating between an sae2::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a sae2::HPH mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC549 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC549.

source_name: DAMC549 chromosomal DNA labeled with Cy5


title: DAMC552 array

description: DAMC552 is an illegitimate diploid from a mating between an mre11::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a mre11::HPH mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC552 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC552.

source_name: DAMC552 chromosomal DNA labeled with Cy5


title: DAMC536 array

description: DAMC536 is an illegitimate diploid from a mating between an sae2::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a sae2::HPH mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC536 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC536.

source_name: DAMC536 chromosomal DNA labeled with Cy5


title: DAMC495 array

description: DAMC495 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and an mre11-H125N mutant version of test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC495 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC495.

source_name: DAMC495 chromosomal DNA labeled with Cy5


title: DAMC551 array

description: DAMC551 is an illegitimate diploid from a mating between an mre11::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a mre11::HPH mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC551 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC551.

source_name: DAMC551 chromosomal DNA labeled with Cy5


title: DAMC539 array

description: DAMC539 is an illegitimate diploid from a mating between an sae2::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a sae2::HPH mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC539 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC539.

source_name: DAMC539 chromosomal DNA labeled with Cy5


title: DAMC547 array

description: DAMC547 is an illegitimate diploid from a mating between an sae2::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and a sae2::HPH mutant version of the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC547 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC547.

source_name: DAMC547 chromosomal DNA labeled with Cy5


title: DAMC485 array

description: DAMC485 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and an mre11-H125N mutant version of test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC485 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC485.

source_name: DAMC485 chromosomal DNA labeled with Cy5


title: DAMC473 array

description: DAMC473 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and an mre11-H125N mutant version of test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC473 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC473.

source_name: DAMC473 chromosomal DNA labeled with Cy5


title: DAMC483 array

description: DAMC483 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and an mre11-H125N mutant version of test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC483 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC483.

source_name: DAMC483 chromosomal DNA labeled with Cy5


title: DAMC479 array

description: DAMC479 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and an mre11-H125N mutant version of test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC479 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC479.

source_name: DAMC479 chromosomal DNA labeled with Cy5

source_name: MS71 chromosomal DNA labeled with Cy3


title: DAMC475 array

description: DAMC475 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and an mre11-H125N mutant version of test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC475 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC475.

source_name: DAMC475 chromosomal DNA labeled with Cy5


title: DAMC476 array

description: DAMC476 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and an mre11-H125N mutant version of test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC476 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC476.

source_name: DAMC476 chromosomal DNA labeled with Cy5


title: DAMC467 array

description: DAMC467 is an illegitimate diploid from a mating between an mre11:HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC467 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC467.

source_name: DAMC467 chromosomal DNA labeled with Cy5


title: DAMC230 array

description: DAMC230 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC230 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC230.

source_name: DAMC230 chromosomal DNA labeled with Cy5


title: KDC_Scer_H20Control2

type: RNA

city: Knoxville

first: Kathleen

last: Cusick

state: TN

address: 676 Dabney Hall

department: Ctr for Environmental Biotechnology

institute: University of Tennessee

description: Single colonies of S. cerevisiae were picked from Yeast Peptone Dextrose (YPD) agar plates (1% yeast extract, 2% peptone, 2% dextrose), inoculated into YPD broth, and incubated at 30 degrees C with 200 rpm agitation for routine maintenance. Cultures were re-inoculated into fresh SC and grown overnight at the same conditions to mid-exponential phase (OD600=1.0), whereupon 400 microliters were removed for exposure to saxitoxin or the water control for 45 minutes prior to extraction.

hyb_protocol: Prior to hybridization, the probe arrays were pre-incubated at 45° C for 10 min with the hybridization buffer (100 mM MES, 1M [Na+], 20mM EDTA, 0.01% Tween-20), which was removed with a sterile pipet tip upon sample loading. Arrays were hybridized for 16 h at 45° C. Hybridization controls included the E. coli bioB, bioC, bioD, and cre genes. Following hybridization, arrays were washed and stained with streptavidin-phycoerythrin for 10 minutes at 25° C, washed, and stained with an anti-streptavidin antibody solution. This was followed by a second stain with streptavidin-phycoerythrin.

scan_protocol: Affymetrix 7G GeneChip high resolution scanner

data_processing: Affymetrix GCOS MAS5.0 Algorithm to obtain detection calls followed by GC-RMA for normalization of signal values

label: Biotin

molecule: total RNA

label_protocol: The Affymetrix one-cycle eukaryotic target labeling assay was used for cDNA synthesis, in vitro transcription, and target labeling.

growth_protocol: Single colonies of S. cerevisiae were picked from Yeast Peptone Dextrose (YPD) agar plates (1% yeast extract, 2% peptone, 2% dextrose), inoculated into YPD broth, and incubated at 30 degrees C with 200 rpm agitation for routine maintenance. Cultures were re-inoculated into fresh SC and grown overnight at the same conditions to mid-exponential phase (OD600=1.0) for exposure to saxitoxin.

extract_protocol: Upon harvesting, cells were stored immediately in liquid nitrogen until RNA extraction. Total RNA was extracted from frozen cell pellets by an initial incubation with equal volumes of pre-warmed phenol:chloroform:isoamylacohol (125:24:1 Ambion) and TES (10 nM Tris pH 7.5, 10 mM EDTA, 0.5% SDS) for 1 h at 65° C with frequent mixing. Samples were centrifuged at 4° C for 20 min, and the aqueous layer extracted and combined with the lysis buffer of the RNEasy kit (Qiagen, Valencia, CA) and 70% ethanol in ratios of 350 microL per 100 microL sample. The remaining steps followed the manufacturer’s protocol. The optional on-column DNase treatment was extended to one hour. Purity and total RNA concentration of each sample were measured with the Nanodrop 1000 (Nanodrop Technologies Inc., Wilmington, DE).

treatment_protocol: Saxitoxin was purchased from the National Research Council Canada (NRCC) (CRM-STX-e). The saxitoxin was lyophilized, concentrated and resuspended in deionized water at a final concentration of 100 microg ml-1. Cultures of S. cerevisiae were grown in 10 mL volumes of YPD to mid-exponential phase, at which point 400 µL from each 10 mL cultures were placed in sterile 2 mL tubes and saxitoxin added to a final concentration of 16 microM (20 microL). An equal volume of deionized water served as the control.


title: DAMC464 array

description: DAMC464 is an illegitimate diploid from a mating between an mre11:HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC464 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC464.

source_name: DAMC464 chromosomal DNA labeled with Cy5


title: DAMC238 array

description: DAMC238 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC238 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC238.

source_name: DAMC238 chromosomal DNA labeled with Cy5


title: DAMC233 array

description: DAMC233 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC233 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC233.

source_name: DAMC233 chromosomal DNA labeled with Cy5


title: DAMC474 array

description: DAMC474 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and an mre11-H125N mutant version of test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC474 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC474.

source_name: DAMC474 chromosomal DNA labeled with Cy5


title: KDC_Scer_H20Control1


title: KDC_Scer_H20Control3


title: DAMC458 array

description: DAMC458 is an illegitimate diploid from a mating between an mre11::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC458 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC458.

source_name: DAMC458 chromosomal DNA labeled with Cy5


title: KDC_Scer_Saxitoxin2


title: DAMC227 array

description: DAMC227 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC227 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC227.

source_name: DAMC227 chromosomal DNA labeled with Cy5


title: DAMC226 array

description: DAMC226 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC226 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC226.

source_name: DAMC226 chromosomal DNA labeled with Cy5


title: DAMC220 array

description: DAMC220 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC220 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC220.

source_name: DAMC220 chromosomal DNA labeled with Cy5


title: DAMC484 array

description: DAMC484 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and an mre11-H125N mutant version of test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC484 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC484.

source_name: DAMC484 chromosomal DNA labeled with Cy5


title: DAMC223 array

description: DAMC223 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC223 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC223.

source_name: DAMC223 chromosomal DNA labeled with Cy5


title: DAMC461 array

description: DAMC461 is an illegitimate diploid from a mating between an mre11::HPH mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC461 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC461.

source_name: DAMC461 chromosomal DNA labeled with Cy5


title: KDC_Scer_Saxitoxin3


title: KDC_Scer_Saxitoxin1


title: DAMC217 array

description: DAMC217 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC217 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC217.

source_name: DAMC217 chromosomal DNA labeled with Cy5


title: DAMC145 array

description: DAMC145 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC145 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC145.

source_name: DAMC145 chromosomal DNA labeled with Cy5

source_name: MS71 chromosomal DNA labeled with Cy3


title: DAMC146 array

description: DAMC146 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC146 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC146.

source_name: DAMC146 chromosomal DNA labeled with Cy5


title: DAMC144 array

description: DAMC144 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC144 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC144.

source_name: DAMC144 chromosomal DNA labeled with Cy5


title: DAMC215 array

description: DAMC215 is an illegitimate diploid from a mating between an mre11-H125N mutant version of the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225alpha (also described in Lemoine et al.).  Genomic DNA from DAMC215 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.  This experiment allows the determination of chromosomal rearrangements in DAMC215.

source_name: DAMC215 chromosomal DNA labeled with Cy5


title: adult male inversion EIn(2L)133.3 vs progenitor cis133.3 replicate 1 dye swap

city: Cambridge

first: Lisa

last: Meadows

address: Downing Street

department: Department of Genetics

country: United Kingdom

institute: University of Cambridge

hyb_protocol: Hybridisation to amino-modified long oligonucleotide microarrays using a Genomic Solutions hybridisation station with the Biosolutions hybridisation buffer (http://www.flychip.org.uk/protocols/hybridisation/)

scan_protocol: Operating instructions for the Genepix 4000B dual laser scanner (http://www.flychip.org.uk/protocols/imaging/)

data_processing: Spot finding and quantitation via the Dapple package

organism: Drosophila melanogaster

label_protocol: Direct Labelling: Up to 50µg total RNA are mixed with anchored oligo (dT)23 (Sigma). The RNA is reverse transcribed using Superscript III (Invitrogen). Cy3-dCTP or Cy5-dCTP (GE Healthcare) are incorporated during cDNA synthesis. (http://www.flychip.org.uk/protocols/gene_expression)

growth_protocol: Flies maintained at 25 degrees C on standard cornmeal agar

extract_protocol: Medium scale RNA extraction using TRIzol (http://www.flychip.org.uk/protocols/gene_expression/)

treatment_protocol: 0-3 day old adult flies were separated into males and females, 20 flies per aliquot, aged at 25C for a further 4 days before transfering to Trizol

tag: gender

value: male

tag: developmental stage

value: adult 4-7 day

tag: tissue

value: whole fly

value: inversion EIn(2L)133.3

value: progenitor cis133.3


title: stamens_C5_rep2

city: Gainesville

first: Andre

last: Chanderbali

state: FL

address: Museum Road and Newell Drive

department: Botany

institute: University of Florida

description: Raw data were read into Bioconductor's Limma package for anlayses

hyb_protocol: In accordance with Agilent's Hybridization protocol

scan_protocol: Agilent's standard Feature Extraction protocol using the Agilent Scanner

data_processing: data represent background corrected and loess normalized signals within arrays

organism: Persea americana

source_name: stamens

label_protocol: In accordance with Agilent's Low Input RNA labeling kit

growth_protocol: Cultivated tree on campus of UF, Gainesville

extract_protocol: Combination of CTAB and QIAGEN Plant RNA extraction protocol as described by Kim et al (2004) Phylogeny and diversification of B-function MADS-box genes in angiosperms: evolution and functional implications of a 260-million-year-old duplication. Am. J. Bot. 91, 2102–2118.

treatment_protocol: frozen in N2

tag: stamens dissected from mature floral buds


title: adult male inversion EIn(2L)133.3 vs progenitor cis133.3 replicate 2


title: DAMC100 (control) array

description: This is a control array.  DAMC100 is a wild-type diploid from a mating between the GAL-POL1 strain described in Lemoine, F. et al. Cell, Vol. 120, 587-598 (2005)  and the test mater 1225a (also described in Lemoine et al.).  Genomic DNA from DAMC100 was competitively hybridized with genomic DNA from the isogenic reference strain MS71.

source_name: DAMC100 chromosomal DNA labeled with Cy5

value: wild-type


title: adult male inversion EIn(1)103.8 vs progenitor cis103.8 replicate 4 dye swap

treatment_protocol: 0-3 day old adult flies were separated into males and females, 20 flies per aliquot, aged at 25C for a further 4 days before transfering males to Trizol

value: whole flies

value: inversion EIn(1)103.8

value: progenitor cis103.8


title: inflor_buds_C5_rep1

source_name: inflorescence buds

tag: pre-emergent whole inflorescence buds


title: adult male inversion EIn(2L)133.3 vs progenitor cis133.3 replicate 4


title: stamens_C5_rep1


title: outer_tepals_C5_rep2

source_name: outer tepals

tag: outer tepals dissected from mature floral buds


title: inflor_buds_C5_rep2


title: outer_tepals_C5_rep1


title: Salivary Gland vs pool 1 of 6

city: Seattle

first: Amit

middle: V

last: Kulkarni

state: WA

address: 401 Terry Ave N

department: Genomics

institute: Rosetta Inpharmatics / Merck Pharmaceuticals

description: Salivary Gland vs pool

hyb_protocol: Microarrays are incubated at 40°C for 48 hours in a rotating carousel.  Hybridizations to custom Agilent microarrays are completed as previously described (Hughes et al.Nat Biotech (2001), 19(4):342-7).  Microarrays are washed to remove non-specific hybridized sample. Afterwards, microarrays are dried in an ozone-free nitrogen chamber.

scan_protocol: Microarrays are scanned using the Agilent LP2 laser scanner. The scanner output is a Tiff file, which contains the quantitative hybridization data from each individual microarray. The Tiff files are then processed using Rosetta custom feature extraction software.

data_processing: Data were processed using the Rosetta Resolver® system. Rosetta's custom feature extraction software performs error modeling before data are loaded into the Resolver system. The Resolver system performs a squeeze operation that combines replicates of the same sequence in an array while applying error weighting. The error weighting consists of adjusting for additive and multiplicative noise. A P-value is generated and propagated throughout the system. The P-value represents the probability that a gene is expressed. The Resolver system allows users to set thresholds, below which genes of a P-value are considered to be significantly expressed. The Resolver system also combines multiple arrays using a squeezing process. If multiple spots reference one sequence, summarization is performed using an error-weighted average as described in Roland Stoughton and Hongyue Dai, Statistical Combining of Cell Expression Profiles. US Patent #6,351,712, February 26, 2002.

organism: Homo sapiens

source_name: Salivary Gland

label_protocol: custom automated version of the aminoallyl MessageAmp II kit from Ambion.

extract_protocol: Qiagen RNeasy spin columns with DNAse treatment

value: Salivary Gland

source_name: pool

value: pool


title: floral_bud_C5_rep2

source_name: floral buds

tag: pre-meiotic floral buds


title: adult male inversion EIn(1)103.8 vs progenitor cis103.8 replicate 2 dye swap


title: adult male inversion EIn(1)101.6 vs progenitor cis101.6 replicate 4 dye swap

value: inversion EIn(1)101.6

value: progenitor cis101.6


title: adult male inversion EIn(1)101.6 vs progenitor cis101.6 replicate 1


title: adult male inversion EIn(2LR)136.4 vs progenitor cis136.4 replicate 3

value: progenitor cis136.4

value: inversion EIn(2LR)136.4


title: adult male inversion EIn(1)103.8 vs progenitor cis103.8 replicate 1


title: adult male inversion EIn(1)101.6 vs progenitor cis101.6 replicate 3


title: adult male inversion EIn(2LR)136.4 vs progenitor cis136.4 replicate 2 dye swap


title: adult male inversion EIn(1)103.8 vs progenitor cis103.8 replicate 3


title: adult male inversion EIn(2LR)136.4 vs progenitor cis136.4 replicate 4 dye swap


title: floral_bud_C5_rep1


title: InnerTepals_C5_rep1

source_name: Inner tepals dissected from mature floral buds

tag: inner tepals dissected from mature floral buds


title: innerTepals_C5_rep2


title: leaves_C5_rep1

source_name: leaves

tag: unexpanded foliar leaves


title: adult male inversion EIn(2LR)136.4 vs progenitor cis136.4 replicate 1


title: carpel_C5_rep2

source_name: carpel dissected from mature floral buds


title: leaves_C5_rep2


title: adult male inversion EIn(2R)137.2/EIn(2LR)138.2 vs progenitor cis137.2/cis138.2 replicate 4 dye swap

treatment_protocol: cis138.2 females were crossed with cis137.2 males. 0-3 day old adult F1 progeny were separated into males and females, 20 flies per aliquot, aged at 25C for a further 4 days before transfering males to Trizol

value: inversion EIn(2R)137.2/EIn(2LR)138.2

value: progenitor cis137.2/cis138.2


title: fruit_C5_rep2

source_name: initiating avocado fruit

tag: initiating avocado fruit


title: adult male inversion EIn(2R)137.2/EIn(2LR)138.2 vs progenitor cis137.2/cis138.2 replicate 1

treatment_protocol: EIn(2LR)138.2 females were crossed with EIn(2R)137.2 males. 0-3 day old adult F1 progeny were separated into males and females, 20 flies per aliquot, aged at 25C for a further 4 days before transfering males to Trizol

value: male


title: fruit_C5_rep1


title: Carpel_C5_rep1

tag: carpel dissected from mature floral buds


title: adult male inversion EIn(2LR)138.2 vs progenitor cis138.2 replicate 5 dye swap

value: adult 4-7 day

value: inversion EIn(2LR)138.2

value: progenitor cis138.2


title: adult male inversion EIn(2R)137.2/EIn(2LR)138.2 vs progenitor cis137.2/cis138.2 replicate 3


title: adult male inversion EIn(2LR)138.2 vs progenitor cis138.2 replicate 2 dye swap


title: adult male inversion EIn(2R)137.2 vs progenitor cis137.2 replicate 6

value: progenitor cis137.2

value: inversion EIn(2R)137.2


title: adult male inversion EIn(2L)133.3 vs progenitor cis133.3 replicate 3 dye swap

value: whole fly

value: inversion EIn(2L)133.3

value: progenitor cis133.3


title: inflor_buds_C3_rep2


title: inflor_buds_C3_rep1


title: adult male inversion EIn(2R)137.2 vs progenitor cis137.2 replicate 5 dye swap

value: whole flies


title: stamens_C3_rep1


title: adult male inversion EIn(2LR)138.2 vs progenitor cis138.2 replicate 1


title: stamens_C3_rep2


title: floral_bud_C3_rep2


title: adult male inversion EIn(2R)137.2 vs progenitor cis137.2 replicate 1


title: outer_tepals_c3_rep2


title: adult male inversion EIn(2L)133.3/EIn(2L)135.1 vs progenitor cis133.3/cis135.1 replicate 5 dye swap

treatment_protocol: EIn(2L)133.3 females were crossed with EIn(2L)135.1/Cy males. 0-3 day old adult F1 progeny were separated into males and females, 20 flies per aliquot, aged at 25C for a further 4 days before transfering non-Cy males to Trizol

treatment_protocol: cis133.3 females were crossed with cis135.1/Cy males. 0-3 day old adult F1 progeny were separated into males and females, 20 flies per aliquot, aged at 25C for a further 4 days before transfering non-Cy males to Trizol


title: floral_bud_C3_rep1


title: outer_tepals_c3_rep1


title: leaves_Cy3_rep2


title: leaves_Cy3_rep1


title: Salivary Gland vs pool 3 of 6

description: Salivary Gland vs pool

value: Salivary Gland

value: pool


title: Salivary Gland vs pool 5 of 6

description: Salivary Gland vs pool


title: adult male inversion EIn(2LR)136.4/EIn(2R)137.2 vs progenitor cis136.4/cis137.2 replicate 2

treatment_protocol: cis137.2 females were crossed with cis136.4 males. 0-3 day old adult F1 progeny were separated into males and females, 20 flies per aliquot, aged at 25C for a further 3 days before transfering to Trizol

value: adult 3-6 day


title: Salivary Gland vs pool 2 of 6

description: Salivary Gland vs pool


title: InnerTepals_Cy3_rep2


title: adult male inversion EIn(2LR)136.4/EIn(2R)137.2 vs progenitor cis136.4/cis137.2 replicate 4


title: InnerTepals_Cy3_rep1


title: adult male inversion EIn(2LR)136.4/EIn(2R)137.2 vs progenitor cis136.4/cis137.2 replicate 1 dye swap

treatment_protocol: EIn(2R)137.2 females were crossed with EIn(2LR)136.4 males. 0-3 day old adult F1 progeny were separated into males and females, 20 flies per aliquot, aged at 25C for a further 3 days before transfering to Trizol


title: adult male inversion EIn(2L)133.3/EIn(2L)135.1 vs progenitor cis133.3/cis135.1 replicate 2 dye swap


title: adult male inversion EIn(2L)133.3/EIn(2L)135.1 vs progenitor cis133.3/cis135.1 replicate 6


title: adult male inversion EIn(2LR)136.4/EIn(2R)137.2 vs progenitor cis136.4/cis137.2 replicate 3 dye swap


title: male progenitor cis133.3 vs female progenitor cis133.3 replicate 3 dye swap

value: female

value: whole body


title: male progenitor cis133.3 vs female progenitor cis133.3 replicate 4


title: fruit_cy3_rep1


title: Carpel_C3_rep2


title: fruit_cy3_rep2


title: male inversion EIn(2L)133.3 vs female inversion EIn(2L)133.3 replicate 4


title: Carpel_C3_rep1


title: male inversion EIn(2L)133.3 vs female inversion EIn(2L)133.3 replicate 3 dye swap


title: adult male, testes, inversion EIn(2L)133.3 vs progenitor cis133.3 replicate 4

label_protocol: Klenow Labelling: Between 3 to 5µg total RNA are reverse transcribed to cDNA [anchored oligo (dT)23 (Sigma), Superscript III (Invitrogen)] and second strand synthesis is then performed [Second strand buffer (Invitrogen), DNA Polymerase I (Invitrogen), RNaseH (New England Biolabs), E. coli DNA ligase (GE Healthcare)] to obtain double stranded DNA. Random primers are annealed to this denatured DNA template and extended by Klenow fragment using the Bioprime DNA Labeling System (Invitrogen) while fluorescent dyes Cy3-dCTP or Cy5-dCTP (GE Healthcare) are incorporated. (http://www.flychip.org.uk/protocols/gene_expression/)

extract_protocol: Small scale RNA extraction using TRIzol (http://www.flychip.org.uk/protocols/gene_expression/)

treatment_protocol: 0-3 day old male adult flies were separated from females, 20 flies per aliquot, aged at 25C for a further 4 days before dissecting out testes and transfering them to Trizol, each testis pair in a separate tube. 8 samples pooled for RNA extraction.

value: testes


title: adult male, testes, inversion EIn(2L)133.3 vs progenitor cis133.3 replicate 2


title: male progenitor cis133.3 vs female progenitor cis133.3 replicate 2


title: Salivary Gland vs pool 6 of 6

description: Salivary Gland vs pool


title: male progenitor cis133.3 vs female progenitor cis133.3 replicate 1 dye swap


title: adult male, testes, inversion EIn(2L)133.3 vs progenitor cis133.3 replicate 3 dye swap


title: male inversion EIn(2L)133.3 vs female inversion EIn(2L)133.3 replicate 2


title: male inversion EIn(2L)133.3 vs female inversion EIn(2L)133.3 replicate 1 dye swap


title: Salivary Gland vs pool 4 of 6

description: Salivary Gland vs pool


title: wild type macrophages stimulated with LPS for 24 hours in presence of EGTA

status: Public on Jun 17 2009

city: Milan

first: Francesca

last: Granucci

address: Piazza della Scienza 2

country: Italy

institute: University of Milano-Bicocca

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 9.5.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 31.4 μg.

hyb_protocol: 10 μg of fragmented cRNAs were hybridized to the standard arrays for 16 hours at 45°C; the arrays were then washed and stained using the fluidics station.

scan_protocol: Arrays were scanned on an Affymetrix GeneChip scanner 3000.

data_processing: Data were processed using Affymetrix Microarray Suite 5.0

label: biotin

organism: Mus musculus

source_name: bone marrow macrophages

label_protocol: Biotinilated cRNA was prepared using GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®).

growth_protocol: Bone marrow cells were cultured for 7 days in IMDM (Euroclone) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-ME (all from Sigma-Aldrich), 10% heat-inactivated FBS (IMDM complete medium), and M-CSF.

treatment_protocol: Macrophages were resuspended in complete IMDM, plated in 10 ml plates (5 millions) cells/plate) and stimulated with smooth LPS (1 μg/ml) in presence of EGTA (2mM). After 24 hours the cells were harvested washed in PBS and processed.

tag: age

value: 6-8 weeks;

value: bone marrow derived macrophages


title: CD14-deficient macrophages stimulated with LPS for 24 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 9.7.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 53.2 μg.

treatment_protocol: Macrophages were resuspended in complete IMDM, plated in 10 ml plates (5 million) cells/plate) and stimulated with LPS (1 μg/ml). After 24 hours cells were harvested, washed in PBS and processed.


title: CD14-deficient macrophages stimulated with LPS for 6 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 9.7.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 67.2 μg.

treatment_protocol: Macrophages were resuspended in complete IMDM, plated in 10 ml plates (5 million) cells/plate) and stimulated with LPS (1 μg/ml). After 6 hours cells were harvested, washed in PBS and processed.


title: wild type macrophages unstimulated

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 9.6.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 53.1 μg.

treatment_protocol: Macrophages were resuspended in complete IMDM, plated in 10 ml plates (5 millions) cells/plate), harvested 24 hours later, washed in PBS and processed.


title: wild type macrophages stimulated with LPS for 24 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 9.5.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 52 μg.

treatment_protocol: Macrophages were resuspended in complete IMDM, plated in 10 ml plates (5 millions) cells/plate) and stimulated with smooth LPS (1 μg/ml). After 24 hours the cells were harvested washed in PBS and processed.


title: CD14-deficient macrophages unstimulated

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 9.7.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 71 μg.

treatment_protocol: Macrophages were resuspended in complete IMDM, plated in 10 ml plates (5 million) cells/plate) harvested 24 hours later, washed in PBS and processed.


title: CD14-deficient dendritic cells stimulated with LPS for 60 hours in presence of thapsigargin

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 8.4.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 20.01 μg.

source_name: bone marrow derived dendritic cells

label_protocol: Biotinilated cRNA was prepared using GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+).

growth_protocol: Bone marrow cells were cultured in IMDM (Euroclone) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-ME (all from Sigma-Aldrich), 10% heat-inactivated FBS (IMDM complete medium), and 10% supernatant of GM-CSF-transduced B16 tumor cells. Fresh medium was added every 3 days. After 7–10 days of culture, cells were analyzed for CD11c expression and were used in assays when >90% were CD11c positive.

treatment_protocol: DCs were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS (1 μg/ml) in presence of thapsigargin (50 nM). After 60 hours, cells were then harvested, washed in PBS and processed.

value: bone marrow derived dendritic cells


title: wild type macrophages stimulated with LPS for 6 hours in presence of EGTA

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 9.7.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 64.7 μg.

treatment_protocol: Macrophages were resuspended in complete IMDM, plated in 10 ml plates (5 millions) cells/plate) and stimulated with smooth LPS (1 μg/ml) in presence of EGTA. After 6 hours the cells were harvested washed in PBS and processed.


title: CD14-deficient dendritic cells stimulated with LPS for 60 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 8.6.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 44.6 μg.

treatment_protocol: DCs were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS (1 μg/ml). After 60 hours, cells were then harvested, washed in PBS and processed.


title: wild type dendritic cells stimulated with LPS for 60 hours in presence of thapsigargin

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 10.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 37.80 μg.

treatment_protocol: DCs were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS (1 μg/ml) in presence of thapsigargin (50 nM) . Cells were harvested 60 hours later, washed in PBS and processed.


title: wild type macrophages stimulated with LPS for 6 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 9.3.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 76 μg.

treatment_protocol: Macrophages were resuspended in complete IMDM, plated in 10 ml plates (5 millions) cells/plate) and stimulated with smooth LPS (1 μg/ml). After 6 hours the cells were harvested washed in PBS and processed.


title: CD14-deficient dendritic cells unstimulated (apoptosis)

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 10.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 38.73 μg.

treatment_protocol: DCs were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) harvested 24 hours later, washed in PBS and processed.


title: wild type dendritic cells stimulated with LPS for 60 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 8.6.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 19 μg.

treatment_protocol: DCs were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS (1 μg/ml). Cells were harvested 60 hours later, washed in PBS and processed.


title: wild type dendritic cells unstimulated (apoptosis)

extract_protocol: Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure.

treatment_protocol: DCs were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and harvested 24 hours later.


title: CD14-deficient dendritic cells stimulated with LPS for 48 hours in presence of thapsigargin

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 9.2.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 23.4 μg.

treatment_protocol: DCs were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS (1 μg/ml) in presence of thapsigargin (50 nM). After 48 hours, cells were then harvested, washed in PBS and processed.


title: CD14-deficient dendritic cells stimulated with LPS for 48 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 10.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 15 μg.

treatment_protocol: DCs were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS (1 μg/ml). After 48 hours, cells were then harvested, washed in PBS and processed.


title: wild type dendritic cells stimulated with LPS for 48 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 9.4.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 31.2 μg.

treatment_protocol: DCs were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS (1 μg/ml). Cells were harvested 48 hours later, washed in PBS and processed.


title: CD14-deficient dendritic cells stimulated with LPS for 6 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.5.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 114 μg.

data_processing: Data were processed using Affymetrix Microarray Suite 5.0.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS for 6 hours. Cells were then harvested, washed in PBS and processed.


title: wild type dendritic cells stimulated with LPS for 48 hours in presence of thapsigargin

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 9.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and MessageAmp III RNA Amplification kit Ambion (+). Biotinylated cRNA was prepared and fragmented according to the Ambion protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 0.3 μg and the obtained cRNA material was 30.3 μg.

treatment_protocol: DCs were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS (1 μg/ml) in presence of thapsigargin (50 nM). Cells were harvested 48 hours later, washed in PBS and processed.


title: CD14-deficient dendritic cells stimulated with LPS for 4 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.3.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 99 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS for 4 hours. Cells were then harvested, washed in PBS and processed.


title: CD14-deficient dendritic cells stimulated with LPS for 24 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.7.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 89 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS for 24 hours. Cells were then harvested, washed in PBS and processed.


title: CD14-deficient dendritic cells stimulated with LPS for 12 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.7.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 106 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS for 12 hours. Cells were then harvested, washed in PBS and processed.


title: CD14-deficient dendritic cells stimulated with LPS for 2 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.7.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 93 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stimulated with smooth LPS for 2 hours. Cells were then harvested, washed in PBS and processed.


title: CD14-deficient dendritic cells unstimulated

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.5.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 108 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), harvested 24 hours later, washed in PBS and processed.


title: wild type dendritic cells stimulated with LPS for 24 hours in presence of EGTA

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 8.7.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 150 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stmulated with smooth LPS (1 μg/ml) in presence of EGTA (2 mM) for 24 hours. Cells were then harvested washed in PBS and processed.


title: wild type dendritic cells stimulated with LPS for 12 hours in presence of EGTA

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 8.8.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 95 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stmulated with smooth LPS (1 μg/ml) in presence of EGTA (2 mM) for 12 hours. Cells were then harvested washed in PBS and processed.


title: wild type dendritic cells stimulated with LPS for 4 hours in presence of EGTA

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.7.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 81 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stmulated with smooth LPS (1 μg/ml) in presence of EGTA (2 mM) for 4 hours. Cells were then harvested washed in PBS and processed.


title: wild type dendritic cells unstimulated EGTA

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.9.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 104 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), harvested 24 hours later washed in PBS and processed.


title: wild type dendritic cells stimulated with LPS for 4 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.7.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 114 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), and treated with smooth LPS (1μg/ml). After 4 hours cells were harvested, washed in PBS and processed


title: wild type dendritic cells stimulated with LPS for 6 hours in presence of EGTA

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.9.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 101 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stmulated with smooth LPS (1 μg/ml) in presence of EGTA (2 mM) for 6 hours. Cells were then harvested washed in PBS and processed.


title: wild type dendritic cells stimulated with LPS for 12 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.7.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 90 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), and treated with smooth LPS (1μg/ml). After 12 hours cells were harvested, washed in PBS and processed


title: wild type dendritic cells unstimulated

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. in this case the RIN value was 9.9.
cRNA Synthesis:
Protocol Used: GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 97 μg.

treatment_protocol: DCs were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/well) and harvested 24 hours later.


title: wild type dendritic cells stimulated with LPS for 24 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.9.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 110 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), and treated with smooth LPS (1μg/ml). After 24 hours cells were harvested, washed in PBS and processed


title: wild type dendritic cells stimulated with LPS for 2 hours

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 10.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 111 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate), and treated with smooth LPS (1μg/ml). After 2 hours cells were harvested, washed in PBS and processed


title: ChIP H3K4 Heart biological replicate 1

status: Public on Jun 16 2009

city: College Station

first: Scott

last: Dindot

state: TX

address: Texas Veterinary Medical Center, 4467 TAMU

department: Veterinary Pathobiology

institute: Texas A&M University

description: ChIP biological replicate 1

hyb_protocol: Labeled MeDIP and ChIP DNA products were processed using the Agilents instructions for two color hybridizations.

scan_protocol: Arrays were scanned on an Axon GenePix 4000A scanner.

data_processing: Images were quantified using the Agilent Feature Extraction Software.

source_name: Heart

label_protocol: 300-500 ng of DNA was diluted to a final volume of 48 ul and 40 ul of random primers was added.  Samples were denatured at 100 C for 10 min.  Ten ul of 10X CTP Nucleotide Mix, 4 ul of Cy3 or Cy5 labeled CTP, and 2 ul of exo Klenow fragment were added to each sample and incubated at 37 C overnight.

extract_protocol: DNA was extracted by Proteinase K digestion followed by phenol chloroform extraction and ethanol precipitation.  Chromatin was isolated using Douncing buffer with homogenization.

value: Heart

tag: antibody

tag: type

value: input


title: wild type dendritic cells stimulated with LPS for 2 hours in presence of EGTA

description:
Total RNA extraction and Quality Controls
Total RNA was extracted following the double extraction protocol: RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (Trizol Invitrogen) followed by a Qiagen RNeasy clean-up procedure. RNA purity was assessed by spectrophotometer (Nanodrop): 260/280 and 260/230 ratios were evaluated. If the 260/280 and 260/230 ratios were < of 1.9, RNA samples were ethanol precipitated. Total RNA integrity was assessed by Agilent Bioanalyzer and the RNA Integrity Number (RIN) was calculated. Only high quality RNAs, with RIN greater than 7 were used in the experiment. In this case the RIN was 9.8.
cRNA Synthesis:
GeneChip® One-Cycle Target Labeling and Control Reagents Kit (Affymetrix®). Biotinylated cRNA was prepared and fragmented according to the Affymetrix protocol. Very strict quality control measures were applied during the target preparation procedures. cRNA before and following fragmentation was analysed by Agilent Bioanalyzer. Typical cRNA products examined on an Agilent 2100 Bioanalyzer have a size range from 500nt to 2500nt with a peak around 1500nt, The expected amount of cRNA obtained should be above 10 times more than the amount of starting total RNA material. Starting RNA material was 3 μg and the obtained cRNA material was 111 μg.

treatment_protocol: Dendritic cells were resuspended in complete IMDM, plated in 10 ml plates (5 million cells/plate) and stmulated with smooth LPS (1 μg/ml) in presence of EGTA (2 mM) for 2 hours. Cells were then harvested washed in PBS and processed.

tag: strain


title: ChIP H3K9 Heart biological replicate 1

description: ChIP biological replicate 1


title: ChIP H3K4  Heart biological replicate 2

description: ChIP biological replicate 2