title: Polarbody_pooled_rep2

status: Public on Oct 12 2011

city: Providence

first: Adrian

last: Reich

state: RI

address: 185 Meeting St

department: MCB

institute: Brown University

data_processing: A custom sequencing primer was used which consisted of the WTA2 primer with the six most 3’ bases of the Illumina sequencing primer. 42bp were sequenced consisting of 9bp of the pseudo-random nonamer from the WTA2 kit and 33bp of the unknown mRNA sequence. The first 9bp and the final base were removed, leaving 32bp sequences to map against the human genome (UCSC hg18) using Illumina’s software, Casava v1.7, allowing only 2 mismatches. Please note, the custom sequencing primer that was used was not as efficient as the standard Illumina sequencing primer. Therefore, there are numerous clusters that have poor quality scores. The raw gene count files (chr##.fa_genes_count.txt) were then loaded into edgeR which normalized the counts using the TMM method (trimmed mean of M values, ###-TMMnormalizedGeneCounts.xls), and these counts were used for all further analyses. The geometric means of the TMM counts of every gene for all oocyte or polar body samples was used to generate a list of the expression levels of all genes and the R package rankedListComparison was used to analyze the expression level lists.

source_name: Polarbody

growth_protocol: Human oocytes were collected from infertility patients undergoing controlled ovarian hyperstimulation for in vitro fertilization (IVF) under standard clinical protocol. Germinal vesicle and MI staged oocytes that were not mature for a clinically indicated intracytoplasmic sperm injection (ICSI) procedure underwent in vitro maturation for 24 hours and were used in the study if they extruded a polar body. Written consent was obtained from all patients to use discarded tissue and oocytes for research, and the study was approved by the institutional review board at Women & Infants Hospital.

extract_protocol: The individual polar bodies and oocytes were processed using the lysis buffer and DNAse 1 from the Ambion Cells-to-Ct Direct kit, followed by reverse transcription and whole transcriptome amplification using the Sigma-Aldrich WTA2 kit. The cDNA was then amplified for 14 cycles in a final volume of 75 µL. In an attempt to maximize cDNA yield, 10μL of the amplified cDNA was added to 65 μL of amplification mix and underwent an additional 15 rounds of amplification specific for cDNA containing the WTA products. The second amplification was combined with the remainder of the first amplification step. Each individual oocyte and polar body were processed in separate reactions and for those samples that were pooled, ten oocytes or polar bodies were pooled together in a common tube after the two rounds of WTA amplification. The 22 oocyte and sibling polar body pairs (44 cells) were split into two replicates of 10 pooled cells and two replicates of a single cells, for a total of 8 samples, 4 oocyte samples and 4 polar body samples. The cDNA was not subjected to any additional shearing and libraries were prepared using the NEBNext DNA Sample Prep Kit with adapters and PCR primers from IDT. The standard protocol was used with starting material of no less than 1.5μg total cDNA with one slight modification. After the ligation of the adapters to the ends of the cDNA molecules, PCR amplification was performed without an intervening gel purification. After the PCR step, gel purification of the completed library was performed and a wide band of 200-450bp was cut from the gel. The library concentration was determined by the Kapa Biosystems qPCR kit and size by Bioanalyzer. The samples were sequenced for 42bp on a GAIIx using a custom sequencing primer consisting of the 6bp most 3’ of the Illumina sequencing primer fused to the WTA2 primer sequence.

treatment_protocol: Patients underwent controlled ovarian hyperstimulation, with either luteal downregulation using a GnRH agonist, pituitary suppression using a GnRH antagonist, or a microdose lupron “flare protocol” consisting of daily lupron injections initiated in the follicular phase with gonadotropins. Oocytes were aspirated by ultrasound guided transvaginal oocyte retrieval 36 hours after injection with recombinant HCG. Four hours after retrieval, all oocytes were mechanically stripped of cumulus cells and immature oocytes not suitable for ICSI were cultured for 20-24 hours in IVM media. Immature oocytes were examined the next day and oocytes that extruded a polar body were used for our study. A total of 22 oocytes and sibling polar bodies were collected and individually processed in this study. All polar body biopsies were performed at 200X magnification after mechanical zona drilling with a polar body biopsy needle. Polar bodies were aspirated into a glass micropipette with an inner diameter of 20µm.

tag: amplification strategy

value: Sigma-Aldrich WTA2


title: Oocyte_pooled_rep2

type: SRA

middle: M

country: USA

library_source: transcriptomic

molecule: total RNA

organism: Homo sapiens

source_name: Oocyte

tag: cell type


title: Oocyte_pooled_rep1


title: ChIP-seq of GFP-tagged ZBTB12 in HEK293 cells

status: Public on Feb 17 2015

city: Montreal

first: Hamed

middle: S

last: Najafabadi

state: QC

address: 740 Dr. Penfield Avenue, Room 7202

department: Human Genetics

country: Canada

institute: McGill University

library_source: genomic

data_processing:
The 3’ end of reads were trimmed so that the final reads were 50 nucleotides long, and the reads were mapped to the human genome build GRCh37 using Bowtie 2 (29), with “--very-sensitive” preset of parameters. This parameter set allows one alignment to be reported for each read that is mapped to multiple positions on the genome, in addition to alignments of uniquely mappable reads. Duplicate reads, which often represent PCR amplification artifacts, were removed using SAMtools.
We performed two rounds of peak-calling: an initial round was performed without an explicit background model of read distributions, in order to identify ChIP experiments with similar experimental/read-mapping biases. We then constructed a background model for each sample, and repeated the peak-calling in order to identify the final set of protein-specific peaks. For the initial round, peaks were identified using MACS v1.4, with fragment length specified based on the results of cross-correlation analysis using SPP package (average 150 bp). Then, the overlap of the identified peaks for each pair of experiments was calculated as the total length of the intersection of peaks divided by the total length of the union of peaks from two experiments. Then, for each experiment x, the top 10 experiments with the highest overlap were identified, so that none of these 10 experiments would represent a biological replicate of experiment x. The reads from these 10 experiments were then randomly sampled, with probabilities proportional to the extent of overlap of each experiment with experiment x, so that a total of 5e+7 reads would be obtained. These reads were then used as the background model in order to perform a second, final round of peak-calling for experiment x, using MACS.
Genome_build: hg19
Supplementary_files_format_and_content: BED files containing peaks and peak summits

molecule: genomic DNA

source_name: HEK293 cells expressing GFP-tagged protein

growth_protocol: HEK293 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and antibiotics. Gateway-compatible entry clones were cloned into the pDEST pcDNA5/FRT/TO-eGFP vector according to the manufacturer's instructions and co-transfected into Flp-In T-REx 293 cells together with the pOG44 Flp recombinase expression plasmid. Cells were selected for FRT site-specific recombination into the genome, following instructions by the manufacturer. Expression of the gene of interest was induced by addition of doxycycline to the culture medium 24 hours prior to harvesting.

extract_protocol:
HEK293 cells were cross-linked for 10 min in 1% formaldehyde. Lysates were sonicated to a DNA fragment length range of 200-300 bp using a Bioruptor (Diagenode). GFP-tagged transcription factors were immunoprecipitated with a polyclonal anti-GFP antibody (ab290, Abcam) and Dynabeads Protein G (Invitrogen). Subsequently, crosslinks were reversed at 65°C over night and bound DNA fragments were purified (EZ-10 Spin Column PCR Product Purification kit, Bio Basic).
Sequencing libraries were constructed using the NEBNext ChIP-Seq Library Prep kit (NEB) according the manufacturer's instructions.

tag: host cell line

tag: transfected with

value: GFP-tagged ZBTB12

tag: uniprotkb id

tag: chip antibody

value: polyclonal anti-GFP antibody

tag: chip antibody vendor

value: Abcam

tag: chip antibody cat. #


title: Oocyte_single_rep2

library_source: transcriptomic single cell


title: ChIP-seq of GFP-tagged ZNF250 in HEK293 cells

value: GFP-tagged ZNF250


title: ChIP-seq of GFP-tagged ZNF675 in HEK293 cells

value: GFP-tagged ZNF675


title: ChIP-seq of GFP-tagged ZNF574 in HEK293 cells

value: GFP-tagged ZNF574


title: ChIP-seq of GFP-tagged ZNF189 in HEK293 cells

value: GFP-tagged ZNF189


title: Oocyte_single_rep1


title: Polarbody_single_rep2


title: ChIP-seq of GFP-tagged ZBTB18 in HEK293 cells

value: GFP-tagged ZBTB18


title: Polarbody_single_rep1


title: ChIP-seq of GFP-tagged KLF10 in HEK293 cells

value: GFP-tagged KLF10


title: Polarbody_pooled_rep1


title: BGA92

status: Public on Sep 29 2010

city: Clay Center

first: Gregory

middle: Paul

last: Harhay

state: NE

department: Genetics and Breeding Research Unit

institute: USDA-ARS-USMARC

description: 16-base (excluding the DpnII restriction site) DGE tag data.

data_processing: Image analysis and basecalling were performed using the Illumina Pipeline v 0.3, where sequence tags were obtained after purity filtering. Fastq files were processed with a custom perl script to create a processed data file with three columns: 1) tag sequence, 2) raw tag counts, and 3) normalized tag counts in tags per million (TPM).

organism: Bos taurus

source_name: bone

extract_protocol:
Sample extraction and total RNA isolation: All bovine tissues were collected following USDA, ARS animal use and care protocols.  Most tissues were snap frozen in liquid N2 immediately after excision.  Exceptions were bone marrow that was separated from bone with a syringe needle and white blood cells collected as a buffy coat by centrifugation (3000 x g, 4oC, 30 min).  Residual red blood cells were removed from the buffy coat by resuspending twice in 140 mM NH4Cl; 17 mM Tris/HCl and collecting the unlysed cells by repeating centrifugation after each resuspension. All samples were stored at -80oC until sonicated or pulverized and then homogenized using a polytron homogenizer for extraction of total RNA with TRIZOL (Molecular Research Center, Cincinnati, OH) using the manufacturer's protocol.  The integrity of the RNA was confirmed a 2100 Bioanalyzer and RNA 6000 Nano-chip (Agilent - Santa Clara, CA).  The samples used had an average RIN value of 8.0 and 28S:18S rRNA ratio of 1.42.

Tag Library Construction and Sequencing: Tag library preparation was performed at Illumina (formerly Solexa, Hayward, CA) using a progenitor version of DGE-Tag Profiling DpnII Sample Prep kit and protocol. In brief, total RNA aliquots (1 or 2 μg) were diluted in 50 uL of nuclease-free H2O and heated at 65oC for 5 min to disrupt secondary structure prior to incubation with magnetic oligo-dT beads to capture the poly-adenylated RNA fraction.  First and second-strand cDNA was synthesized and bead-bound cDNA was subsequently digested with DpnII to retain a cDNA fragment from the 3’-most GATC to the poly(A)-tail.  Unbound cDNA fragments were washed away prior to ligation with GEX DpnII adapter to the 5’ end of the bead-bound digested cDNA fragments. This adapter contains a restriction site for MmeI that cuts 17 bp downstream from the DpnII site.  After subsequent digestion with MmeI, 21 bp tags starting with the DpnII recognition sequence were recovered from the beads and dephosphorylated prior to phenol/chloroform extraction.  Then, a second adapter (GEX adapter 2) was ligated onto the 3’ end of the cDNA tag at the MmeI cleavage site.  The adapter-ligated cDNA tags were enriched by a 15-cycle PCR amplification using Phusion polymerase (Finnzymes) and primers complementary to the adapter sequences.  The resulting fragments were purified by excision from a 6% polyacrylamide TBE gel. The DNA was eluted from the gel debris with 1× NEBuffer 2 by gentle rotation for 2 h at room temperature. Gel debris were removed using Spin-X Cellulose Acetate Filter (2 ml, 0.45 µm) and the DNA was precipitated by adding 10 µl of 3 M sodium acetate (pH 5.2) and 325 µl of ethanol (–20°C), followed by centrifugation at 14,000 r.p.m. for 20 min. After washing the pellet with 70% ethanol, the DNA was resuspended in 10 µl of 10 mM Tris–HCl, pH8.5 and quantified with a Nanodrop 1000 spectrophotometer.  Sequencing using Solexa/Illumina Whole Genome SequencerCluster generation was performed after applying 4 pM of each sample to the individual lanes of the Illumina 1G flowcell. After hybridization of the sequencing primer to the single-stranded products, 18 cycles of base incorporation were carried out on the 1G analyzer according to the manufacturer's instructions.

tag: tissue

value: bone

tag: sex

value: M

tag: developmental stage

value: 180 d fetus

tag: breed

value: Hereford

tag: animal source

value: WGS Offspring


title: BGA96

source_name: all pituitary

value: pituitary


title: BGA98

source_name: lymph-body cavity

value: lymph-body cavity


title: BGA94

source_name: kidney

value: kidney


title: BGA9

source_name: left ovary w/10s/2m/1l

value: ovary

value: F

value: juvenile


title: BGA90

source_name: salivary gland

value: salivary gland


title: BGA79

source_name: jejunum

value: jejunum


title: BGA76

source_name: duodenum

value: duodenum


title: BGA88

source_name: ventricle

value: ventricle


title: BGA64


title: BGA77

source_name: ileum

value: ileum


title: BGA80

source_name: reticulum

value: reticulum


title: BGA89

source_name: lung (redo of lja27)

value: lung


title: BGA82

source_name: thymus

value: thymus

value: M


title: BGA91

source_name: adrenal

value: adrenal

value: 180 d fetus

value: Hereford


title: BGA78

source_name: omasum

value: omasum

value: WGS Offspring


title: BGA8

source_name: uterine cross-section w/ glandular region

value: uterus with glandular region


title: BGA85

source_name: hippocampus

value: hippocampus


title: BGA81

source_name: rumen

value: rumen


title: BGA65

source_name: large intestine

value: large intestine

value: F

value: juvenile


title: BGA63

value: duodenum


title: BGA74

source_name: abomasum

value: abomasum


title: BGA66

source_name: adrenal medulla

value: adrenal medulla


title: BGA7

source_name: skin/back

value: skin/back


title: BGA62

value: ileum


title: BGA71

source_name: testes

value: testes

value: M


title: BGA61

source_name: oviduct

value: oviduct


title: BGA60

value: Hereford


title: BGA70

source_name: umbilical cord

value: umbilical cord

value: 180 d fetus


title: BGA5

source_name: thalamus

value: thalamus

value: WGS Offspring


title: BGA55

source_name: nasal epithelium

value: nasal epithelium


title: BGA53

source_name: liver

value: liver


title: BGA72

source_name: vas deferens

value: vas deferens


title: BGA57

source_name: cecum

value: cecum

value: F

value: juvenile


title: BGA83

source_name: thyroid

value: thyroid


title: BGA49

source_name: temporal cortex

value: temporal cortex


title: BGA47

source_name: frontal cortex

value: frontal cortex


title: BGA54

source_name: adrenal cortex

value: adrenal cortex


title: BGA58

source_name: white fat

value: white fat


title: BGA52


title: BGA46

source_name: medulla oblongata

value: medulla oblongata


title: BGA32

source_name: midbrain

value: midbrain

value: Hereford


title: BGA48

source_name: retina

value: retina


title: BGA45

source_name: pons

value: pons


title: BGA22

source_name: uterus-intercaruncular

value: uterus-intercaruncular

value: adult

value: WGS Cow


title: BGA39

source_name: cheek lymph nodes

value: cheek lymph nodes

value: WGS Offspring


title: BGA3

source_name: cerebral cortex

value: cerebral cortex

value: F


title: BGA21

source_name: spleen-internal piece

value: spleen

value: juvenile


title: BGA171

source_name: preconfluent perirenal adipocytes

tag: sample type

value: cell line

value: preconfluent perirenal adipocytes

value: TAMU


title: BGA173

source_name: 90 d lactating gland

value: 90 d lactating gland

value: Holstein

value: BARC


title: BGA38

source_name: outer tongue surface

value: outer tongue surface

value: Hereford


title: BGA41

source_name: tongue muscle

value: tongue muscle


title: BGA51

source_name: diaphragm

value: diaphragm


title: BGA18

source_name: rumen

value: rumen


title: BGA33

source_name: spinal cord

value: spinal cord


title: BGA19

source_name: mammary/parenchyma

value: mammary/parenchyma

value: WGS Offspring


title: BGA50

source_name: oral mucosa

value: oral mucosa

value: F


title: BGA37

source_name: mesenteric lymph nodes

value: mesenteric lymph nodes

value: juvenile


title: BGA2

source_name: basal ganglia

value: basal ganglia


title: BGA172

source_name: differentiated perirenal adipocytes

value: cell line

value: differentiated perirenal adipocytes

value: TAMU


title: BGA16

source_name: hypothalamus

value: hypothalamus


title: BGA4

source_name: hippocampus

value: hippocampus


title: BGA20

source_name: placenta above cotlydon

value: placenta above cotlydon

value: M

value: 180 d fetus

value: Hereford


title: BGA23

source_name: kidney-internal piece

value: kidney


title: BGA157

source_name: biceps femoris

value: biceps femoris

value: Guernsey

value: BARC


title: BGA149

source_name: infraspinitus

value: infraspinitus


title: BGA154

source_name: subq fat

value: subcutaneous fat

value: adult

value: Angus

value: USMARC


title: BGA134

source_name: sternomandibuilaris satellite muscle cells

value: cell line

value: LARRL


title: BGA133

source_name: white fat

value: white fat

value: WGS Offspring


title: BGA25

source_name: stomach omasum

value: stomach omasum

value: F

value: juvenile


title: BGA13

source_name: skeletal muscle near ceasarian opening

value: skeletal muscle near ceasarian opening

value: Hereford

value: WGS Cow


title: BGA150

source_name: longissimus

value: longissimus

value: M


title: BGA148

source_name: longissimus dorsi

value: longissimus dorsi

value: Guernsey

value: BARC


title: BGA137

source_name: cerebellum

value: cerebellum


title: BGA142

source_name: small intestine/d7 hc

value: small intestine

value: Angus


title: BGA156

source_name: marbling fat

value: marbling fat

value: adult

value: USMARC


title: BGA129

source_name: 3rd eyelid necrotic/outside tumor

value: 3rd eyelid necrotic/outside tumor

value: Line 1 Cow


title: BGA152

value: infraspinitus


title: BGA130

source_name: healthy 3rd eyelid

value: healthy 3rd eyelid

value: F


title: BGA126

source_name: coronary band

value: coronary band

value: 180 d fetus

value: Hereford

value: WGS Offspring


title: BGA151

value: biceps femoris

value: M


title: BGA138

source_name: anterior pituitary

value: anterior pituitary

value: juvenile


title: BGA153

source_name: semitendinosous

value: semitendinosous

value: Angus


title: BGA135

source_name: nucleated blood cells

value: nucleated blood cells

value: adult

value: WGS Cow


title: BGA146

source_name: fat pad of mammary

value: mammary fat pad

value: Holstein

value: BARC


title: BGA105

source_name: liver

value: liver


title: P. furiosus-peroxide shock-t=30-rep 2-3

status: Public on Feb 25 2010

type: RNA

city: Athens

first: Michael

middle: W. W.

last: Adams

state: GA

address: 120 East Green St.

department: Molecular Biology and Biochemistry

institute: University of Georgia

description: biological replicate 2-3

hyb_protocol: Sample preparation steps for microarray hybridization:  Before proceeding make sure hybridizer has been prepared and ready to accept labeled cDNA samples (i.e. tubing in correct wash bottles, o-rings patted into chambers, treated slides in place and correct program loaded).   Make sure each labeled sample is dissolved in an appropriate volume of nuclease free water.  Add a ratio of volumes of 6.5:1 of Sigma ArrayHyb hybridization buffer to a mixture of each sample with nuclease free water. Make sure that the combined volumes of all samples does not exceed 150uL.  Combine the labeled cDNA samples to form a total volume of 150 uL of labeled cDNA.  Denature the combined sample for 1 minute at 90C.  Inject sample into hybridization chamber. The following is a standard hybridization method that seems to work well for this procedure: 14 hours at 65C  2 wash cycles at 40C with 2X SSC w/0.1% Tween-20 2 cycles at 35C wash with 0.2X SSC w/0.1% Tween-20 2 wash cycles at 30C with0.2X SSC

scan_protocol: Images were aquired with a scanarray 5000 (PE) with 4 lasers. Channels were balanced using the line scan feature and by balancing the overall signals without the use of external standards Description         Scatterplots were used to verify overall quality of the data making sure both channels are equal. Data processing         Signals for each channel were obtained using Gleams (Nutec) and background were subtracted using the median local intensity around the spot.

data_processing: Fluorescent intensities were background subtracted and log2 ratios are presented

label: alexa 647

organism: Pyrococcus furiosus DSM 3638

source_name: peroxide shock t=0 min

label_protocol: In a sterile microfuge tube add 40uL of 27 OD/mL Random 9-mer Primer (Stratagene). Speed vacuum the centrifuge tube with the primer until no liquid is left. The RNA to be added in this procedure is assumed to be 2 ug/uL. What matters is that the amount of RNA has to be 50 ug in order for this procedure to work. Add all reagents in the order that they appear to a centrifuge with dried primer in it. Reagent volume 25 ul RNA (2 ug/uL)   10 uL Nuclease free water  Total volume 35uL Flick to mix and then spin lightly (approx 4,000 rpm’s). Incubate @ 60C for 5 min, after incubation immediately inject RNase block, mix and allow to equilibrate to R.T. Flick to mix and then spin lightly (approx 4,000 rpm). 1 uL RNase Block  2.5uL 25 mM MgCl2  10uL 10X RT Buffer  10uL 10mM dCTP, dGTP, dATP/0.5 mM TTP 20 uL 2 mM aminoallyl dUTP  5.0 uL Stratascript RT (50 U/mL) Total volume 83.5uL Incubate for 30 mins @ 37C 3.0 uL Stratascript RT (50 U/mL)  Total volume 86.5uL Incubate for 30 mins @ 37C. 11uL 1 M NaOH Incubate for 8 minutes @ 60C 20uL 2.4 M MOPS @ pH 4.0. Total volume 117.5uL  First purification:  Check pH of sample before going to purification step. The pH MUST BE LOWER THAN < 7.0. In order for complete labeled cDNA recovery. Heat denature sample at 90C for 1-2 minutes before loading onto spin columns.  Mix RT-PCR products with 500 uL Qiagen Binding Buffer and filter through spin columns (~4,000 rpm).  Wash out centrifuge tube with an extra 100uL of Binding Buffer and spin down (~4,000 rpm).  Wash spin column 2 times with 500uL of 80% ethanol/buffered water provided in Qiagen purification kit. The last spin should be faster (~14,000 rpm’s) to make sure all ethanol is removed.  Elute labeled cDNA 3 times with 20 uL of nuclease free water into sterile 1.5 mL microfuge tubes (allow the first 20uL to soak into spin-column for 5 min before centrifugation). The desired labeled cDNA is in the WATER ELUTION. Speed vacuum sample until dry (not too dry, i.e. don’t leave in speed vacuum for over 3 hours). Fluorescent Labeling:  Add 5.0uL of  nuclease free water and 3 uL of 300 mM sodium bicarbonate to the dried cDNA sample.   Dissolve and mix thoroughly each Alexa dye to be used with 5uL DMSO. Typically Alexa dyes contain two applications in each vial. It is recommended to label two sample sets at the same time.  Mix 2.5 uL of dye with prepared cDNA sample  Incubate in the dark (i.e.: wrap in tin foil) for 1 hour @ R.T.  Second purification:  Check pH of 488 Alexa sample ONLY (the other dyes will not give conclusive readings) sample before going to purification step. The pH MUST BE LOWER THAN < 7.0!!! in order for complete labeled cDNA recovery. If pH is not correct adjust using 1.0 μL of 2.4 M MOPS @ pH 4.0.  Mix RT-PCR products with 500 uL Qiagen Binding Buffer and filter through spin columns (~4,000 rpm).  Wash out centrifuge tube with an extra 100 uL of Binding Buffer and spin down (~4,000 rpm).  Wash spin column 2 times with 500uL of 80% ethanol/buffered water provided in Qiagen purification kit. The last spin should be faster (~14,000 rpm’s) to make sure all ethanol is removed.  Elute labeled cDNA 3 times with 20 ul of nuclease free water. Speed vacuum sample until dry (not too dry, i.e. don’t leave in speed vacuum for over 1 hour)

growth_protocol: Cells were grown in a 20 l custom build fermentor on artificial seawater with 5 g/l maltose as the carbon source, with 0.5 g/l yeast extract to provide additional nutrients. Cells were harvested at a density of about 5 exp07 cells/ml

extract_protocol: Collect ~2 liters of culture and spin samples at 6000 rpm’s for 15 minutes cooled to 4C. Once finished with first spin, decant.  Make absolutely sure the last drop of medium is removed. Spin down left over liquid after decanting at 3000 rpm’s for 5 minutes cooled to 4C.  Keep samples on ice until phenol is added.  Using only 1.0 mL of ice cold 3% NaCl combine 2 cell pellets into a 15 mL Corning tube (should result in 3 tubes, each with 1 mL of solution).  Add 3.0 mL of Lysis buffer and 0.35 mL 3.0 M Na-acetate pH 5.0 and 3 mL acid phenol (5:1).  Mix well, but do not vortex.  Spin for 20 min, 3000 rpm, at room temperature in the tabletop centrifuge.  Take carefully the upper aqueous phase and transfer to a clean falcon tube.  To the supernatant add 3.0 mL of acid phenol, mix well and spin 20 min, 3000 rpm, at room temperature in the tabletop centrifuge.  Take carefully the upper aqueous phase and transfer to a clean falcon tube.  To the supernatant add 3.0 mL of chloroform, mix well spin 20 min, 3000 rpm, at room temperature in the tabletop centrifuge.  Make 0.9 mL aliquots in 2.0 mL Eppendorf tube and precipitate the RNA with 1.0 mL isopropanol.  Store in -80C. RNA Isolation Protocol: Part 2  Allow samples to equilibrate to 4C in an ice bath for ~ 1 hour.  Spin down RNA for 20 min at 14,000 rpm, then pipette off supernatant, re-spin the tubes for another 5 min at 14,000 rpm and pipette off last drop of liquid.  Take up RNA in 100 uL RNase free water and add 500 ul Lysis buffer and 70 uL 3.0 M Na-acetate pH 5.0, mix well.  To the supernatant add 600 uL acid phenol and mix, shake well but do not vortex.  Centrifuge samples for 20 min at 14,000 rpm (room temperature).  Take the water (top) phase and extract this with 600 uL chloroform.  Centrifuge samples for 20 min at 14,000 rpm (room temperature).  Take the water phase (top layer) and add 600 uL isopropanol (RNase free) store sample in freezer, at -20C, for 1-2 hours .  Centrifuge samples for 20 min at 14,000 rpm (room temperature).  Pipette off supernatant and wash the pellet with 1.0 mL cold 70% ethanol (Rnase free).  Centrifuge samples for 20 min at 14,000 rpm (room temperature).  Completely remove supernatant, re-spin sample for 2 min at 14,000 rpm and pipette off the rest of the supernatant.  Air dry the RNA pellet for 5 min. The color of the pellet should be white.  Dissolve the RNA pellet in ~100 uL absolute RNase free water.  Measure RNA concentration by diluting 5 uL of concentrated RNA solution into 495 uL of RNase free water (concentration might be between 0.1 –10 ug/μL). Assay diluted sample by UV 260 λ and 280 λ (RNA OD of 1 = 40 ug/mL).  Once concentrated RNA sample concentration is determined the sample can be diluted or aliquoted then precipitated to for long term storage if necessary.  If the sample is to be stored for a long time it is better to precipitate RNA as follows: Precipitate the aliquot with 0.1 volume 3.0 M Na-acetate and 3 volumes ethanol then store at -80C.

treatment_protocol: 0.5 mM H2O2 was added to P. furiosus cells growing in the fermentor and rapidly stirred. After 30 min a 2 l sample was drawn

tag: treatment

value: P. furiosus control

label: alexa 546

source_name: peroxide shock t=30

value: P. furiosus peroxide shock t=30 min


title: BGA11

source_name: bone marrow cells

value: bone marrow

value: 180 d fetus

value: Hereford


title: BGA140

source_name: small intestine/d3 hc

value: small intestine

value: F


title: P. furiosus-peroxide shock-t=30-rep 2-2

description: biological replicate 2-2


title: P. furiosus-peroxide shock-t=30-rep 3-3

description: biological replicate 3-3


title: P. furiosus-peroxide shock-t=30-rep 2-1

description: biological replicate 2-1


title: P. furiosus-peroxide shock-t=30-rep 1-3

description: biological replicate 1-3


title: BGA110

source_name: basal ganglia

value: basal ganglia

value: M

value: WGS Offspring


title: BGA108

source_name: ovary right side

value: ovary

value: juvenile


title: BGA36

source_name: pineal gland

value: pineal gland


title: BGA143

source_name: peyer's patch/d7 hc

value: peyer's patch

value: Angus


title: P. furiosus-peroxide shock-t=30-rep 1-1

description: biological replicate 1-1


title: BGA14

source_name: whole testes

value: whole testes

value: adult

value: BAC Bull


title: P. furiosus-peroxide shock-t=30-rep 3-1

description: biological replicate 3-1

value: P. furiosus control

value: P. furiosus peroxide shock t=30 min


title: P. furiosus-peroxide shock-t=30-rep 1-2

description: biological replicate 1-2


title: P. furiosus-peroxide shock-t=30-rep 3-2

description: biological replicate 3-2


title: Pyrococcus furiosus sulfur kinetics t=60 22806 71306

status: Public on May 25 2007

description: Scatterplots were used to verify overal quality of the data making sure both channels are equal.

hyb_protocol: Sample preparation steps for microarray hybridization: • Before proceeding make sure hybridizer has been prepared and ready to accept labeled cDNA samples (i.e. tubing in correct wash bottles, o-rings patted into chambers, treated slides in place and correct program loaded). If you are not sure talk to Dr. Gerti Schut or Scott Brehm. • Make sure each labeled sample is dissolved in an appropriate volume of nuclease free water. • Add a ratio of volumes of 6.5:1 of Sigma ArrayHyb hybridization buffer to a mixture of each sample with nuclease free water. Make sure that the combined volumes of all samples does not L!!!exceed 150 • Combine the labeled cDNA samples to form a total volume of L of labeled cDNA.150 • Denature the combined sample for 1 minute at 90 C. • Inject sample into hybridization chamber. • The following is a standard hybridization method that seems to work well for this procedure: 14 Chours at 65 C with 2X SSC w/0.1% Tween-202 wash cycles at 40 2 wash C with 0.2X SSC w/0.1% Tween-20cycles at 35 C with2 wash cycles at 30  0.2X SSC

scan_protocol: Images were aquired with a scanarray 5000 (PE) with 4 lasers. Channels were balanced using the line scan feature and by balancing the overal signals without the use of external standards

data_processing: Signals for each channel were obtained using Gleams (Nutec) and were background substrated using the median local intensity around the spot.

organism: Pyrococcus furiosus

label_protocol:
Labeling Steps:
L of 27 OD/mL RandomIn a sterile microfuge tube add 40  9-mer Primer (Stratagene, see appendix). Speed vacuum the centrifuge tube with the primer until no liquid is left.
The RNA to be added in this procedure is g inuL. What matters is that the amount of RNA has to be 50 g/assumed to be 2  order for this procedure to work.
Add all reagents in the order that they appear to a centrifuge
with dried primer in it.
Reagent Volume
RNA (2 LuL) 25 g/
LNuclease free water 10
LTotal volume 35
Flick to mix and spin lightly (approx 4,000 rpm’s)
C for 15 mins, afterIncubate @ 60  incubation immediately inject RNase block, mix and allow to equilibrate to R.T. Flick to mix and then spin lightly (approx 4,000 rpm)
RNase Block 1 L
L25 mM MgCl2 2.5
L10X RT Buffer 10
10 mM dCTP, dGTP, LdATP/0.5 mM TTP 10
L2 mM aminoallyl dUTP 20.0
Stratascript RT (50 LU/mL) 5.0
LTotal volume 83.5
CIncubate for 30 mins @ 37
LStratascript RT (50 U/mL) 3.0
LTotal volume 86.5
Incubate Cfor 30 mins @ 37
L1 M NaOH 11
CIncubate for 8 minutes @ 60
L2.4 M MOPS @ pH 4.0 20
LTotal volume 117.5
First purification:
• Check pH of sample before going to purification step. The pH MUST BE LOWER THAN < 7.0!!! in order for complete labeled cDNA recovery.
• C for 1-2 minutes before loading onto spinHeat denature sample at 90  column!
L Qiagen Binding Buffer and filter• Mix RT-PCR products with 500  through spin columns (~4,000 rpm).
• Wash out centrifuge tube with an extra L of Binding Buffer and spin down (~4,000 rpm).100
• Wash spin column 2 L of 80% ethanol/buffered water provided in Qiagen purificationtimes with 500  kit. The last spin should be faster (~14,000 rpm’s) to make sure all ethanol is removed.
L of nuclease free water into• Elute labeled cDNA 3 times with 20  L to soak into spin-columnsterile 1.5 mL microfuge tubes (allow the first 20  for 5 mins before centrifugation). The desired labeled cDNA is in the WATER ELUTION. Speed vacuum sample until dry (not too dry, i.e. don’t leave in speed vacuum for over 3 hours).
Fluorescent Labeling:
L of nuclease• Add 5.0  L of 300 mM sodium bicarbonate to the dried cDNAfree water and 3.0  sample.
L of• Dissolve and mix thoroughly each Alexa dye to be used with 5  DMSO. Typically Alexa dyes contain two applications in each vial. It is recommended to label two sample sets at the same time.
L of dye• Mix 2.5  with prepared cDNA sample.
• Incubate in the dark (i.e.: wrap in tin foil) for 1 hour @ R.T.
Second purification:
• Check pH of 488 Alexa sample ONLY (the other dyes will not give conclusive readings) sample before going to purification step. The pH MUST BE LOWER THAN < 7.0!!! in order for complete labeled cDNA recovery. If pH is not correct adjust using 1.0 μL of 2.4 M MOPS @ pH 4.0.
L Qiagen Binding Buffer and filter• Mix RT-PCR products with 500  through spin columns (~4,000 rpm).
• Wash out centrifuge tube with an extra L of Binding Buffer and spin down (~4,000 rpm).100
• Wash spin column 2 L of 80% ethanol/buffered water provided in Qiagen purificationtimes with 500  kit. The last spin should be faster (~14,000 rpm’s) to make sure all ethanol is removed.
L of nuclease free water.• Elute labeled cDNA 3 times with 20  Speed vacuum sample until dry (not too dry, i.e. don’t leave in speed vacuum for over 1 hour).

growth_protocol: Cells were grown in a 20 l custom build fermentor on artificial seawater with 5 g/l maltose as the carbon source, with 0.5 g/l yeast extract to provide additional nutrients. Cells were harvested at a density of about 0.8 exp08 cells/ml

extract_protocol:
RNA Isolation Protocol: Part 1
• Collect ~2 liters of culture and spin samples at 6000 rpm’s for 15 minutes cooled to 4 ˚C.
• Once finished with first spin, decant.
• Make absolutely sure the last drop of medium is removed. Spin down left over liquid after decanting at 3000 rpm’s for 5 minutes cooled to 4 ˚C.
• Keep samples on ice until phenol is added!
• Using only 1.0 mL of ice cold 3% NaCl combine 2 cell pellets into a 15 mL Corning tube (should result in 3 tubes, each with 1 mL of solution).
• Add 3.0 mL of Lysis buffer and 0.35 mL 3.0 M Na-acetate pH 5.0 and 3 mL acid phenol (5:1).
• Mix well, but do not vortex.
• Spin for 20 min, 3000 rpm, at room temperature in the tabletop centrifuge.
• Take carefully the upper aqueous phase and transfer to a clean falcon tube.
• To the supernatant add 3.0 mL of acid phenol, mix well and spin 20 min, 3000 rpm, at room temperature in the tabletop centrifuge.
• Take carefully the upper aqueous phase and transfer to a clean falcon tube.
• To the supernatant add 3.0 mL of chloroform, mix well spin 20 min, 3000 rpm, at room temperature in the tabletop centrifuge.
• Make 0.9 mL aliquots in 2.0 mL Eppendorf tube and precipitate the RNA with 1.0 mL isopropanol.
• Store in -80˚C.
RNA Isolation Protocol: Part 2
• Allow samples to equilibrate to 4 ˚C in an ice bath for ~ 1 hour.
• Spin down RNA for 20 min at 14,000 rpm, then pipette off supernatant, re-spin the tubes for another 5 min at 14,000 rpm and pipette off last drop of liquid.
• Take up RNA in 100 μL RNase free water and add 500 μl Lysis buffer and 70 μL 3.0 M Na-acetate pH 5.0, mix well.
• To the supernatant add 600 μL acid phenol and mix, shake well but do not vortex.
• Centrifuge samples for 20 min at 14,000 rpm (room temperature).
• Take the water (top) phase and extract this with 600 μL chloroform.
• Centrifuge samples for 20 min at 14,000 rpm (room temperature).
• Take the water phase (top layer) and add 600 μL isopropanol (RNase free) store sample in freezer, at -20˚C, for 1-2 hours .
• Centrifuge samples for 20 min at 14,000 rpm (room temperature).
• Pipette off supernatant and wash the pellet with 1.0 mL cold 70% ethanol (make sure it is Rnase free).
• Centrifuge samples for 20 min at 14,000 rpm (room temperature).
• Completely remove supernatant, re-spin sample for 2 min at 14,000 rpm and pipette off the rest of the supernatant.
• Air dry the RNA pellet for 5 min. The color of the pellet should be white.
• Dissolve the RNA pellet in ~100 μL absolute RNase free water.
• Measure RNA concentration by diluting 5 μL of concentrated RNA solution into 495 μL of RNase free water (concentration might be between 0.1 –10 μg/μL). Assay diluted sample by UV 260 λ and 280 λ (RNA OD of 1 = 40 μg/mL).
• Once concentrated RNA sample concentration is determined the sample can be diluted or aliquoted then precipitated to for long term storage if necessary.
• If the sample is to be stored for a long time it is better to precipitate RNA as follows:
Precipitate the aliquot with 0.1 volume 3.0 M Na-acetate and 3 volumes ethanol then store at -80˚C.

treatment_protocol: cells were used as reference

tag: whole cells

label: alexa 488

label_protocol: same culture as channel 1

growth_protocol: same culture as channel 1

extract_protocol: same culture as channel 1

treatment_protocol: 2 g/l sulfur flower was added to the fermentor and rapidly stirred in. After 10 min a 2 l sample was drawn


title: Pyrococcus furiosus sulfur kinetics t=60 22806 70706


title: Pyrococcus furiosus sulfur kinetics t=20 22806 71306


title: Pyrococcus furiosus sulfur kinetics t=60 20806 71706

label: alexa 594


title: Pyrococcus furiosus sulfur kinetics t=60 20806 62406


title: Pyrococcus furiosus sulfur kinetics t=20 20806 71706


title: Pyrococcus furiosus sulfur kinetics t=30 22806 72106


title: Pyrococcus furiosus sulfur kinetics t=30 20806 71706


title: Pyrococcus furiosus sulfur kinetics t=30 20806 71706s


title: Pyrococcus furiosus sulfur kinetics t=20 20806 21606


title: Pyrococcus furiosus sulfur kinetics t=30 22806 62006


title: Pyrococcus furiosus sulfur kinetics t=20 22806 70706


title: Pyrococcus furiosus sulfur kinetics t=10 22806 72106


title: BGA158

source_name: kidney capsule fat

value: kidney capsule fat

value: Guernsey

value: BARC


title: Control heart NH4

status: Public on Nov 21 2004

city: Napoli

first: Lucio

last: Nitsch

address: Via Pansini 5

department: Biologia e Patologia Cellulare e Molecolare

country: Italy

institute: Unversity of Naples Federico II

description:
The heart tissue was explanted from a human fetus with normal karyotype and without atrio-ventricular septal defects, following therapeutic abortion. The posterior half representing all the four heart chambers was dissected by a longitudinal cut. Total RNA was extracted and PolyA+ mRNA was obtained to prepare biotinilated target cRNA according to Affymetrix protocols (http://www.affymetrix.com/support/technical/manuals/expression_manual.affx). Target cRNA was hybridized to Affymetrix HG-U133A chip using an Affymetrix GeneChip Instrument System according to the procedures described at http://www.affymetrix.com/support/downloads/manuals/expression_s2_manual.pdf.
Image data were analyzed using the Affymetrix expression analysis software. The Affymetrix software Microarray Suite (MAS 5.0) assigned to each probe set of the chip an average difference value corresponding to the expression level of the gene it represents and an absolute call that indicates whether the transcript is present in the sample. Data sets were scaled to a total targeted fluorescence of 100.
Keywords = Heart gene expression

data_processing: Affymetrix CEL files were imported into GeneSping 7.2 software (Agilent Technologies Inc.) and normalized by MAS5 algorithm

label: Biotin

source_name: Fetal heart at 21 weeks of gestation

label_protocol: In vitro transcription was performed to obtain biotin labeled cRNA according to Affymetrix protocols described at http://www.affymetrix.com/support/technical/manuals.affx

extract_protocol: Total RNA was extracted by Trizol

value: heart from fetus with Down syndrome and septal defects.

tag: age

value: 21 gestation weeks


title: Pyrococcus furiosus sulfur kinetics t=30 22806 70706


title: Down syndrome heart DH5

description:
The heart tissue was explanted from a human fetus with chromosome 21 trisomy without atrio-ventricular septal defects, following therapeutic abortion. The posterior half representing all the four heart chambers was dissected by a longitudinal cut. Total RNA was extracted and PolyA+ mRNA was obtained to prepare biotinilated target cRNA according to Affymetrix protocols (http://www.affymetrix.com/support/technical/manuals/expression_manual.affx). Target cRNA was hybridized to Affymetrix HG-U133A chip using an Affymetrix GeneChip Instrument System according to the procedures described at http://www.affymetrix.com/support/downloads/manuals/expression_s2_manual.pdf.
Image data were analyzed using the Affymetrix expression analysis software. The Affymetrix software Microarray Suite (MAS 5.0) assigned to each probe set of the chip an average difference value corresponding to the expression level of the gene it represents and an absolute call that indicates whether the transcript is present in the sample. Data sets were scaled to a total targeted fluorescence of 100.
Keywords = Down syndrome
Keywords = heart gene expression

hyb_protocol: according to Affymetrix protocols described at http://www.affymetrix.com/support/technical/manuals.affx

source_name: Fetal heart at 19 weeks of gestation

value: 19 gestation weeks


title: Pyrococcus furiosus sulfur kinetics t=10 22806 62006

hyb_protocol: Sample preparation steps for microarray hybridization: •	Before proceeding make sure hybridizer has been prepared and ready to accept labeled cDNA samples (i.e. tubing in correct wash bottles, o-rings patted into chambers, treated slides in place and correct program loaded).  If you are not sure talk to Dr. Gerti Schut or Scott Brehm. •	Make sure each labeled sample is dissolved in an appropriate volume of nuclease free water. •	Add a ratio of volumes of 6.5:1 of Sigma ArrayHyb hybridization buffer to a mixture of each sample with nuclease free water.  Make sure that the combined volumes of all samples does not exceed 150 uL!!! •	Combine the labeled cDNA samples to form a total volume of 150 uL of labeled cDNA. •	Denature the combined sample for 1 minute at 90 C. •	Inject sample into hybridization chamber. •	The following is a standard hybridization method that seems to work well for this procedure: 14 hours at 65 C 2 wash cycles at 40 C with 2X SSC w/0.1% Tween-20 2 wash cycles at 35 C with 0.2X SSC w/0.1% Tween-20 2 wash cycles at 30 C with 0.2X SSC

label_protocol:
Labeling Steps:
In a sterile microfuge tube add 40 uL of 27 OD/mL Random 9-mer Primer (Stratagene, see appendix).  Speed vacuum the centrifuge tube with the primer until no liquid is left.
The RNA to be added in this procedure is assumed to be 2 g/uL.  What matters is that the amount of RNA has to be 50 g in order for this procedure to work.
Add all reagents in the order that they appear to a centrifuge
with dried primer in it.
Reagent	Volume
RNA (2 g/uL)	25 uL
Nuclease free water	10 uL
Total volume	35 uL
Flick to mix and spin lightly (approx 4,000 rpm’s)

Incubate @ 60 C for 15 mins, after incubation immediately inject RNase block, mix and allow to equilibrate to R.T.  Flick to mix and then spin lightly (approx 4,000 rpm)

RNase Block 	1 uL

25 mM MgCl2	2.5 uL
10X RT Buffer	10 uL
10 mM dCTP, dGTP, dATP/0.5 mM TTP	10 uL
2 mM aminoallyl dUTP	20.0 uL
Stratascript RT (50 U/mL)	5.0 uL
Total volume	83.5 uL

Incubate for 30 mins @ 37 C

Stratascript RT (50 U/mL)	3.0 uL
Total volume	86.5 uL

Incubate for 30 mins @ 37 C

1 M NaOH	11 uL

Incubate for 8 minutes @ 60 C

2.4 M MOPS @ pH 4.0	20 uL
Total volume	117.5 uL
First purification:
•	Check pH of sample before going to purification step.  The pH MUST BE LOWER THAN < 7.0!!! in order for complete labeled cDNA recovery.
•	Heat denature sample at 90 C for 1-2 minutes before loading onto spin column!
•	Mix RT-PCR products with 500 uL Qiagen Binding Buffer and filter through spin columns (~4,000 rpm).
•	Wash out centrifuge tube with an extra 100 uL of Binding Buffer and spin down (~4,000 rpm).
•	Wash spin column 2 times with 500 uL of 80% ethanol/buffered water provided in Qiagen purification kit.  The last spin should be faster (~14,000 rpm’s) to make sure all ethanol is removed.
•	Elute labeled cDNA 3 times with 20 uL of nuclease free water into sterile 1.5 mL microfuge tubes (allow the first 20 uL to soak into spin-column for 5 mins before centrifugation).  The desired labeled cDNA is in the WATER ELUTION.  Speed vacuum sample until dry (not too dry, i.e. don’t leave in speed vacuum for over 3 hours).
Fluorescent Labeling:
•	Add 5.0 uL of nuclease free water and 3.0 uL of 300 mM sodium bicarbonate to the dried cDNA sample.
•	Dissolve and mix thoroughly each Alexa dye to be used with 5 uL of DMSO.  Typically Alexa dyes contain two applications in each vial.  It is recommended to label two sample sets at the same time.
•	Mix 2.5 uL of dye with prepared cDNA sample.
•	Incubate in the dark (i.e.: wrap in tin foil) for 1 hour @ R.T.
Second purification:
•	Check pH of 488 Alexa sample ONLY (the other dyes will not give conclusive readings) sample before going to purification step.  The pH MUST BE LOWER THAN < 7.0!!! in order for complete labeled cDNA recovery.  If pH is not correct adjust using 1.0 μL of 2.4 M MOPS @ pH 4.0.
•	Mix RT-PCR products with 500 uL Qiagen Binding Buffer and filter through spin columns (~4,000 rpm).
•	Wash out centrifuge tube with an extra 100 uL of Binding Buffer and spin down (~4,000 rpm).
•	Wash spin column 2 times with 500 uL of 80% ethanol/buffered water provided in Qiagen purification kit.  The last spin should be faster (~14,000 rpm’s) to make sure all ethanol is removed.
•	Elute labeled cDNA 3 times with 20 uL of nuclease free water.  Speed vacuum sample until dry (not too dry, i.e. don’t leave in speed vacuum for over 1 hour).

extract_protocol:
RNA Isolation Protocol: Part 1
•	Collect ~2 liters of culture and spin samples at 6000 rpm’s for 15 minutes cooled to 4 ˚C.
•	Once finished with first spin, decant.
•	Make absolutely sure the last drop of medium is removed.  Spin down left over liquid after decanting at 3000 rpm’s for 5 minutes cooled to 4 ˚C.
•	Keep samples on ice until phenol is added!
•	Using only 1.0 mL of ice cold 3% NaCl combine 2 cell pellets into a 15 mL Corning tube (should result in 3 tubes, each with 1 mL of solution).
•	Add 3.0 mL of Lysis buffer and 0.35 mL 3.0 M Na-acetate pH 5.0 and 3 mL acid phenol (5:1).
•	Mix well, but do not vortex.
•	Spin for 20 min, 3000 rpm, at room temperature in the tabletop centrifuge.
•	Take carefully the upper aqueous phase and transfer to a clean falcon tube.
•	To the supernatant add 3.0 mL of acid phenol, mix well and spin 20 min, 3000 rpm, at room temperature in the tabletop centrifuge.
•	Take carefully the upper aqueous phase and transfer to a clean falcon tube.
•	To the supernatant add 3.0 mL of chloroform, mix well spin 20 min, 3000 rpm, at room temperature in the tabletop centrifuge.
•	Make 0.9 mL aliquots in 2.0 mL Eppendorf tube and precipitate the RNA with 1.0 mL isopropanol.
•	Store in -80˚C.
RNA Isolation Protocol: Part 2
•	Allow samples to equilibrate to 4 ˚C in an ice bath for ~ 1 hour.
•	Spin down RNA for 20 min at 14,000 rpm, then pipette off supernatant, re-spin the tubes for another 5 min at 14,000 rpm and pipette off last drop of liquid.
•	Take up RNA in 100 μL RNase free water and add 500 μl Lysis buffer and 70 μL 3.0 M Na-acetate pH 5.0, mix well.
•	To the supernatant add 600 μL acid phenol and mix, shake well but do not vortex.
•	Centrifuge samples for 20 min at 14,000 rpm (room temperature).
•	Take the water (top) phase and extract this with 600 μL chloroform.
•	Centrifuge samples for 20 min at 14,000 rpm (room temperature).
•	Take the water phase (top layer) and add 600 μL isopropanol (RNase free) store sample in freezer, at -20˚C, for 1-2 hours .
•	Centrifuge samples for 20 min at 14,000 rpm (room temperature).
•	Pipette off supernatant and wash the pellet with 1.0 mL cold 70% ethanol (make sure it is Rnase free).
•	Centrifuge samples for 20 min at 14,000 rpm (room temperature).
•	Completely remove supernatant, re-spin sample for 2 min at 14,000 rpm and pipette off the rest of the supernatant.
•	Air dry the RNA pellet for 5 min.  The color of the pellet should be white.
•	Dissolve the RNA pellet in ~100 μL absolute RNase free water.
•	Measure RNA concentration by diluting 5 μL of concentrated RNA solution into 495 μL of RNase free water (concentration might be between 0.1 –10 μg/μL).  Assay diluted sample by UV 260 λ and 280 λ (RNA OD of 1 = 40 μg/mL).
•	Once concentrated RNA sample concentration is determined the sample can be diluted or aliquoted then precipitated to for long term storage if necessary.
•	If the sample is to be stored for a long time it is better to precipitate RNA as follows:
Precipitate the aliquot with 0.1 volume 3.0 M Na-acetate and 3 volumes ethanol then store at -80˚C.

treatment_protocol: Cells were used as reference

label_protocol: same as channel 1

extract_protocol: same as channel 1


title: Down syndrome heart DH3

scan_protocol: according to Affymetrix protocols described at http://www.affymetrix.com/support/technical/manuals.affx

source_name: Fetal heart at 20 weeks of gestation

value: 20 gestation weeks


title: Control heart NH5


title: Control heart NH2


title: Down syndrome heart DH4


title: Control heart NH3


title: Pyrococcus furiosus sulfur kinetics t=10 20806 021606

source_name: whole cells

tag: Hyperthermophilic archaeon

source_name: alexa 488

tag: same as channel 1


title: Control heart NH1


title: Down syndrome heart DH1

description:
The heart tissue was explanted from a human fetus with chromosome 21 trisomy and without atrio-ventricular septal defects, following therapeutic abortion. The posterior half representing all the four heart chambers was dissected by a longitudinal cut. Total RNA was extracted and PolyA+ mRNA was obtained to prepare biotinilated target cRNA according to Affymetrix protocols (http://www.affymetrix.com/support/technical/manuals/expression_manual.affx). Target cRNA was hybridized to Affymetrix HG-U133A chip using an Affymetrix GeneChip Instrument System according to the procedures described at http://www.affymetrix.com/support/downloads/manuals/expression_s2_manual.pdf.
Image data were analyzed using the Affymetrix expression analysis software. The Affymetrix software Microarray Suite (MAS 5.0) assigned to each probe set of the chip an average difference value corresponding to the expression level of the gene it represents and an absolute call that indicates whether the transcript is present in the sample. Data sets were scaled to a total targeted fluorescence of 100.
Keywords = Down syndrome
Keywords = heart gene expression


title: Pyrococcus furiosus sulfur kinetics t=30 20806 62406


title: Pyrococcus furiosus sulfur kinetics t=10 20806 71706


title: BGA101

source_name: sme

value: stalk median eminence

value: M

value: 180 d fetus

value: Hereford

value: WGS Offspring


title: Down syndrome heart DH6