title: Drosophila melanogaster02

status: Public on Dec 31 2009

type: RNA

city: Lexington

first: Subba

middle: Reddy

last: Palli

state: KY

address: S-225 Agr. Sci. Ctr. N

department: Entomology

country: USA

institute: University of Kentucky

description: Gene expression data from DMSO treated and JH treated for 3, 6 and 12hr.

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the Affymetrix GCS 3000 scanner.

data_processing: The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.

label: Biotin

molecule: total RNA

organism: Drosophila melanogaster

source_name: Drosophila L57 DMSO-treated

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

growth_protocol: Drosophila L57 cells were grown and maintained at 26 ± 1°C in 25-cm2 cell culture flasks using HyQ CCM3 medium (HyClone, Logan, UT) with 5% FBS (heat inactivated, suitable for insect cells from Sigma).

extract_protocol: Total RNA from L57 cells (JH III-treated and DMSO-treated) was extracted with RNeasy Total RNA Isolation kit from QIAGEN (Valencia, CA) according to the manufacturer's instructions.

treatment_protocol: Cultured Drosophila L57 cells were exposed to 1 uM JH III for 3, 6 and 12 h.

tag: Drosophila L57 DMSO vs juvenile hormone III treated (3, 6 12h)


title: Alanine

city: Detroit

first: Laila

middle: M

last: Poisson

state: MI

address: 1 Ford Place

department: Biostatistics and Research Epidemiology

institute: Henry Ford Health Systems

description: Samples 3 and 4 are technical replicates

hyb_protocol: 825ng of Cy3 and Cy5 labelled aRNA were hybridized competitively for 17 hrs in a 65 C hybridization oven (G2545A, Agilent) set to 10 rpm in a final concentration of 1X GEx Hybridization Buffer HI-RPM, according to the manufacturer's recommnded protocol (Two-Color Microarray-Based Gene Expression Analysis, Agilent). Arrays were washed according to the manufacturer's recommended protocol including the Stabilization and Drying Solution step (Two-Color Microarray-Based Gene Expression Analysis, Agilent).

scan_protocol: Arrays were scanned at 5um resolution on an Agilent DNA Microarray Scanner (G2505B, Agilent) using the default settings for 4x44k format two-color arrays.

data_processing: Images were auto gridded, analyzed and data extracted using Agilent Feature Extraction Software (Version 9.5.1.1). Spot values were normalized using the default linear-lowess normalization. Samples M5a and M5b were isolated from the same patient and normalized values were averaged before data analysis.

organism: Homo sapiens

source_name: untreated PrEC cells

label_protocol: One ug of total RNA was reverse transcripted to cDNA using T7 Promoter Primer and MMLV-RT. Then the cDNA was converted to aRNA using T7 RNA polymerase, which simultaneously amplifies target material and incorporates cyanine 3- or cyanine 5-labeled CTP (Two-Color Microarray-Based Gene Expression Analysis Protocol, Agilent).

growth_protocol: The cells are grown in Prostate Epithelial Cell Medium (Lonza Walkersville Inc. Walkersville, MD, Cat No CC-0310) supplemented with  Bovine Pitutary extract, Hydrocortisone, human Epidermal Growth factor, Transferrin, Insulin, retinoic Acid, Triiodothyronine, and GA-1000 as per manufacturers instruction.

extract_protocol: Total RNA was isolated with Trizol (Invitrogen Cat. No. 15596-026, Carlsbad, CA), according to the manufacturer's instructions. Total RNA was further purified using the Qiagen RNAeasy Mini Kit (Cat. No. 74104) according to the manufacturer's instructions.

treatment_protocol: The cells are grown to 60-70% confluence and treated with 50 uM concentration of either alanine, leucine, glycine or sarcosine (Sigma, St Louis, MO), all resuspended in water for a period of 24h.

tag: PrEC cells from Lonza (Cat No CC-0310)

source_name: Alanine treated PrEC cells, rep1

tag: 50 uM alanine added to PrEC cells


title: Drosophila melanogaster01

source_name: Drosophila embryos L57 DMSO-treated (control)

treatment_protocol: Cultured Drosophila L57 Cells were exposed to 1 uM JHIII for 3, 6 and 12 hr.


title: E10.5 ko.2

status: Public on Dec 28 2009

city: Shanghai

first: Ming

last: Hu

address: 197 Ruijin Road II

country: China

institute: Shanghai Institute of Hematology

description: total RNA from mouse embryo yolk sacs, 4 individual yolk sacs of each genotype were pooled as sample.

hyb_protocol: standard Affymetrix procedures

scan_protocol: standard Affymetrix procedures

data_processing: Gene expression was quantified by robust multi-array analysis (RMA)

label: biotin

organism: Mus musculus

source_name: E10.5 embryo yolk sacs

label_protocol: The One-Cycle Target Labeling and Control Reagent kit (Affymetrix #900493) and the protocols optimized by Affymetrix were used to prepare biotinylated cRNA (from 5μg of total RNA) for microarray hybridization.

extract_protocol: Total RNA was extracted from individual yolk sacs (n = 4 per group) using RNAzol (Campro Scientific, Veenendaal, The Netherlands) and glass beads according to the manufacturer's instructions.

tag: Strain


title: E10.5 wt.1


title: E9.0 wt.2

source_name: E9.0 embryo yolk sacs


title: E9.0 wt.3


title: E10.5 ko.1


title: Drosophila melanogaster121

source_name: Drosophila L57 JH III 12h


title: Drosophila melanogaster62

source_name: Drosophila L57 Cells JHIII 6h


title: Drosophila melanogaster122

source_name: Drosophila L57 Cells JH III 12h


title: E9.0 wt.1


title: Drosophila melanogaster32

source_name: Drosophila L57 JHIII 3h


title: E9.0 ko.2


title: Drosophila melanogaster31

source_name: Drosophila L57 Cells JHIII 3h


title: mouse Spleen_female#3_exposed to Pb

status: Public on Dec 21 2009

city: Rensselaer

first: Sridar

middle: V

last: Chittur

state: NY

address: One Discovery Drive, CRC 342G

department: Center for Functional Genomics

institute: SUNY-University at Albany

description: female Balb/c spleen 21 days exposed to 0.1mM Pb since gd8

hyb_protocol: Codelink standard protocol

scan_protocol: Codelink standard protocol, Axon 4000B, 600V (100%), 635nm

data_processing: Codelink v4.2 image processing software, median normalized

source_name: mouse spleen

label_protocol: Codelink standard protocol

extract_protocol: Qiagen RNeasy midi protocol with on-column Dnase step

tag: BALB/c, female 21 days (exposed to 0.1mM Pb since gd8)


title: Alanine (duplicate)

source_name: Alanine treated PrEC cells, rep2


title: mouse Spleen_female#1_exposed to Pb

tag: BALB/c, female, 21 days (exposed to 0.1mM Pb since gd8)


title: Sarcosine (duplicate)

description: Samples 1 and 2 are technical replicates

source_name: Sarcosine treated PrEC cells, rep2

tag: 50 uM sarcosine added to PrEC cells


title: Sarcosine

source_name: Sarcosine treated PrEC cells, rep1


title: E10.5 wt.3

extract_protocol:
Total RNA was extracted from individual yolk sacs (n = 4 per group) using RNAzol (Campro Scientific, Veenendaal, The Netherlands) and glass beads according to the manufacturer's instructions.
biotin


title: Drosophila melanogaster61


title: mouse Spleen_female#3_control

description: female Balb/c spleen 21 days control

tag: BALB/c, female, 21 days


title: E10.5 ko.3


title: E10.5 wt.2


title: E9.0 ko.3


title: E9.0 ko.1


title: SCGB3A2 treatment replicate 2

status: Public on Dec 26 2009

city: Yokohama

first: Reiko

last: Kurotani

state: Kanagawa

country: Japan

institute: Yokohama City University School of Medicine

description: Gene regulation by SCGB3A2 in Nkx2-1-null lungs.  Culture media with or without SCGB3A2 were changed in two days.

hyb_protocol: Using FairPlay Microarray Labeling Kit (Catalog #252002, Stratagene, La Jolla, CA).

scan_protocol: Scanned on a GenePix 4000A [54243] .According to GenePix protocol.

data_processing: LOWESS normalized, background subtracted VALUE data obtained from log2 of processed Red signal/processed Green signal. Agilent software was used.

source_name: RNA from 4 day-organ cultured Nkx2-1-null lungs with SCGB3A2 treatment.

label_protocol: RNAs labeled with Cy3 and Cy5 (GE Healthcare Life Sciences, Piscataway, NJ) using FairPlay Microarray Labeling Kit (Catalog #252002, Stratagene, La Jolla, CA).

growth_protocol: Fetal lungs (E11.5) were cultured in DMEM/F12 containing 10%FBS on a 0.4mm pore membrane, which was placed on the top of steel wire mesh in an organ culture dish.

extract_protocol: RNAs were amplified using MessageAmp aRNA Kit (Cat #1750, Ambion, Austin, TX)

treatment_protocol: Two hundred fifty ng/ml of Trx-His-tagged recombinant SCGB3A2 protein was added into DMEM/F12 growth medium.

tag: Four day-organ cultured Nkx2-1-null lungs at E16.5 with SCGB3A2 (250ng/ml)

source_name: RNA from 4 day-organ cultured Nkx2-1-null lungs without SCGB3A2 treatment.

tag: Four day-organ cultured Nkx2-1-null lungs at E16.5 without SCGB3A2


title: SCGB3A2 treatment replicate 4


title: SCGB3A2 treatment replicate 1


title: SCGB3A2 treatment replicate 3


title: mouse Spleen_female#2_exposed to Pb


title: sDf36

status: Public on Dec 18 2009

type: genomic

city: Vancouver

first: Martin

middle: Richard

last: Jones

state: BC

address: Room 419 NCE Building 2125 East Mall

department: Medical Genetics

country: Canada

institute: University of British Columbia

scan_protocol: The scanner used was an Axon Scanner (Model # 4000B) . See Selzer, R.R., T.A. Richmond, N.J. Pofahl, R.D. Green, P.S. Eis, P. Nair, A.R. Brothman, and R.L. Stallings. 2005. Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH. Genes Chromosomes Cancer 44: 305-319.

data_processing: Segmentation analysis based on the DNACopy package; Segmentation GFF file; Genome annotation GFF file

molecule: genomic DNA

organism: Caenorhabditis elegans

source_name: whole animal, mixed stage; Strain: BC1784

label_protocol: Cy3 and Cy5 dye-labeled random 9mers (TriLink BioTechnologies, Inc.) were diluted to 1 O.D./42 µl of buffer containing 0.125M Tris-HCl (pH 8.0), 0.125M MgCl2, 1.75 µl/ml β-Mercaptoethanol. Mutant DNA samples were labelled with Cy3 and the wild type DNA sample (VC196) was labelled with Cy5. 1 µg of genomic DNA was added to each random 9mer buffer solution, denatured at 95 ºC and then chilled on ice in 0.2 ml PCR tubes. 10 µl of 50X dNTP mixture (1X TE buffer, 10 mM each of dATP, dCTP, dGTP and dTTP), 8 µl DI water and 100 U Klenow fragment (exo-) was added to each tube and mixed well with a pipet. Samples were centrifuged and incubated at 37 ºC for 2 hours. 10 µl 0.5 M EDTA was added and mixed well to stop the labeling reaction. DNA was precipitated by adding 11.5 µl 0.5 M NaCl and 110 µl isopropanol, vortexing, incubating in the dark for 10 minutes at room temperature, and centrifuging at 12,000X G for 10 minutes. The supernatant was removed and the DNA pellet was washed with 500 µl 80% ethanol. After centrifugation at 12,000X G for 2 minutes, the supernatant was removed and the pellet was dried in a SpeedVac on low heat for 5 minutes before being rehydrated in 25 µl DI water. DNA concentration was measured using a spectrophotometer.

growth_protocol: Nematodes were grown at 200C on 150mm NGM agar plates seeded with Escherichia coli strain OP50.

extract_protocol: Animals were harvested and treated with a standard protienase K digestion. Samples were extracted using a standard phenol/chloroform protocol, treated with RNAse A for 1 hour and subjected to a second round of phenol/chloroform extraction. DNA was resuspended in water and quantified.

source_name: whole animal, mixed stage; Strain: BC2842


title: mouse Spleen_female#2_control

description: female Balb/c spleen 21 days control

tag: BALB/C, female, 21 days


title: sDf44

hyb_protocol: See Selzer, R.R., T.A. Richmond, N.J. Pofahl, R.D. Green, P.S. Eis, P. Nair, A.R. Brothman, and R.L. Stallings. 2005. Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH. Genes Chromosomes Cancer 44: 305-319.

source_name: whole animal, mixed stage; Strain: BC3472


title: sDf52

source_name: whole animal, mixed stage; Strain: BC3541


title: mouse Spleen_female#1_control

description: female Balb/c spleen 21 days control


title: sDf32

source_name: whole animal, mixed stage; Strain: BC1554


title: sDf38

source_name: whole animal, mixed stage; Strain: BC3463


title: sDf45

source_name: whole animal, mixed stage; Strain: BC3473


title: sDf30

source_name: whole animal, mixed stage; Strain: BC1785


title: sDf39

source_name: whole animal, mixed stage; Strain: BC3462


title: Vaccine cells alone d7-S7_280705

status: Public on Dec 17 2009

city: Heidelberg

first: Maximilian

last: Aigner

address: Im Neuenheimer Feld 280

country: Germany

institute: Deutsches Krebsforschungszentrum (DKFZ)

description: In total, seven samples were treated in parallel throughout the whole protocol. Especially to avoid inter-experimental fluctuations, the image acquisition was performed with all seven membranes at once, i.e. the membranes were placed into one (!!!) plastic sheet protector and one (!!!) image was taken from all (!!!) membranes.

hyb_protocol: Hybridization and Chemolumiscent Detection was performed using the Chemoluminescent Detection Kit (D-01; version 1.5, 3/4/2005), purchased from SuperArray Bioscience Corporation. The detailed protocol is given: A. Pre-hybridization: The array membrane was pre-wet for 5 min by adding 5 ml of deionized water to the GEArray hybridization tube. Then 2 ml of 60 °C-warm GEAhyb Hybridization Solution (H-01) were added to the tube (after having discarded the water) and vortexed briefly. The tube was pre-hybridized in an hybridization oven at 60 °C for 2 hours with continuous agitation (10 rpm). B. Hybridization: 10 µg of biotin-labeled cRNA target were added to 0,75 ml of pre-warmed GEAhyb Hybridization Solution (this target hybridization mix was kept at 60 °C until use). The pre-hybridization solution was discarded from the hybridization tube and the target hybridization mix added. The hybridization itself was carried out overnight at 60 °C with continuous agitation (10 rpm). C. Washing: After hybridization, the target hybridization mix was discarded and the membrane washed with 5 ml of Wash Solution 1 for 15 min at 60 °C with 30 rpm agitation. Wash Solution 1 consisted of 2 X SSC and 1 % (w/v) SDS. Wash Solution 1 was then discarded. Afterwards the membrane was washed with 5 ml of Wash Solution 2 for exactly 15 min at 60 °C with 30 rpm agitation. Wash Solution 2 was then immediately discarded and the membrane (in its hybridization tube) allowed to cool to RT. Wash Solution 2 consisted of 0,1 X SSC and 0,5 % (w/v) SDS. Wash Solution 2 is a high stringency wash buffer. Its salt concentration is very critical for good microarray results. Excessive washing of the membrane may result in a loss of signal. D. Chemoluminescent Detection (carried out at RT): D1. The membrane was blocked by adding 2 ml GEAblocking Solution Q and incubation for 40 min with continuous agitation (30 rpm). D2. Alkaline phosphatase-streptavidin conjugate (AP-SA) was diluted 1:8.000 into 1 X Buffer F (diluted from a 5 X stock solution). 2 ml of this AP-SA dilution were added to the membrane (after having discarded the GEAblocking Solution Q) and incubated for exactly 10 min with continuous but gentle agitation (10 rpm). D3. The membrane was washed four times with 4 ml 1 X Buffer F for 5 min with gentle agitation (10 rpm). D4. Finally, the last wash was discarded and the membrane rinsed twice with 3 ml Buffer G. D5. Detection was performed by addition of 1 ml CDP-Star chemoluminescent substrate to the hybridization tube and incubation for 5 min at RT with gentle agitation (10 rpm). Using forceps, the membrane was taken out of its hybridization tube and its corners touched to a piece of filter paper to remove excess of CDP-Star solution. The membrane was placed into a plastic sheet protector and bubbles smoothed out.

scan_protocol: The image acquisition was performed with the help of a cooled (-30 °C) CCD camera (Lumi-Imager F1TM; Roche, Mannheim, Germany; resolution: 1024 x 1280 pixel; pixel size: 16 x 16 µm^2) and an exposure time of 10 min. The image was saved as a 16 bit grayscale, TIFF-formatted image. The LumiAnalyst software (v. 3.1.0) was used.

data_processing:
The image data were processed with the use of the automated, web-based GEArray Expression Analysis Suite (v. 1.1).
Briefly, the process involved the following steps:
1. The TIFF-formatted image of the array membrane was loaded into the Image Data Extraction Applet (IDEA).
2. A cropping frame was set to delineate the borders of the array membrane (the cropped area was defined as closely around the actual array spots as possible).
3. Using the Image/Settings tool, the threshold, brightness and cotrast of the image were adjusted manually to the individual settings of the specific computer monitor (and to the individual perception of the grayscale colors by the user). This helped for the subsequent adjustment of the grid (see step 4).
4. A grid was aligned to the spots on the array membrane using the Autoalign Tool. Nonetheless, all grid spots were counter-checked by eye and corrected manually if necessary. The raw spot intensities were measured automatically.
5. The Grid Readout (containing the raw signal data) was saved.
6. The analysis of the raw data for subsequent evaluation was performed in two steps:
6a. Background correction: the Global Background Correction tool was selected, since it evaluates an overall density value per area within the grid (and also within the cropping frame) but outside of grid capture.
6b. Normalization: after background correction, all array data values were normalized using the housekeeping gene RPS27A (ribosomal protein 27A) which was present in triplicate (ID_REFs 1,2 and 13). The normalized data were obtained using the following mathematical transformation:
Nx = (Rx – b) / (((Rn1 – b) + (Rn2 – b) + (Rn3 – b))/3)
where Nx is the normalized value of the gene x, Rx is the raw value of the gene x, Rn1/Rn2/Rn3 are the three independent raw values for RPS27A, and b is the background (GBC value from step 6a).
7. The background-corrected and normalized array data were subsequently used for pairwise comparison studies.

source_name: Naive human T cells, isolated from peripheral blood mononuclear cells (PBMCs) of a healthy donor.

label_protocol:
Total RNA was biotin-labeled using the TrueLabeling-AMPTM Linear RNA Amplification Kit (GA-010; version 1.3, 3/4/2005), purchased from SuperArray Bioscience Corporation.
This kit is designed to linearly amplify and label antisense RNA (aRNA, also known as cRNA: complementary RNA) for hybridization to the Oligo GEArray from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. cDNA synthesis from extracted total RNA:
A1. Preparation of the Annealing Mixture:
2,0 µg of total RNA,
1,0 µl of TrueLabeling Primer (G1),
0,5 µl of CD45 primer (specially designed by SuperArray Bioscience Corporation) and
RNase-free water
were combined in a sterile PCR tube for a final volume of 10 µl.
The CD45 primer was needed in the reaction to facilitate target labeling for alternative spliced CD45 transcripts. Because of lack of appropriate testing material for alternative CD45 transcripts, SuperArray Bioscience Corporation was unable to confirm the effectiveness of this primer in their QC experiment. Therefore, no guaranty was given for the performance of this primer.
A2. Preparation of cDNA Synthesis Reaction:
To the 10 µl annealing mixture, the following components were added (final volume: 20 µl):
4 µl RNase-free water,
4 µl 5 X cDNA Synthesis Buffer (G3),
1 µl RNase Inhibitor (RI) and
cDNA Synthesis Enzyme Mix (G2).
The reaction was carried out by incubation for 50 minutes at 42 °C.
B. cRNA Synthesis, Labeling and Amplification from cDNA:
To the 20 µl 37 °C-warm cDNA synthesis reaction, the following components were added in the following order (final volume: 40 µl)
16 µl 2,5 X RNA Amplification Buffer (G4),
2 µl Biotin-16-uridine-5’-triphosphate (Roche, # 11 388 908 910) and
2 µl Amplification Enzyme Mix (G5).
The reaction was carried out by incubation overnight (18 hours) at 37 °C.
C. cRNA Purification:
The purification of the labeled and amplified cRNA was performed using the ArrayGrade cRNA Cleanup Kit (GA-012; version 1.3, 3/4/2005).
C1. The cRNA sample was prepared by (i) adding on ice 60 µl of RNase-free water for a final volume of 100 µl (the reaction was then transferred to a 1.5 ml RNase-free microcentrifuge tube), (ii) adding 350 µl Lysis & Binding Buffer (G6) and (iii) immediately adding 350 µl of RT 100 % ethanol.
C2. The cRNA was loaded onto a supplied Spin Column and centrifuged for 30 sec at 8.000 g (binding of the cRNA to the Spin Column). The flow through was discarded.
C3. 600 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then another 200 µl of Washing Buffer working solution were applied and centrifuged for 3 min at 11.000 g (second wash).
C4. cRNA was eluted from the Spin Column by placing the Spin Column onto a fresh 1.5 ml RNase-free microcentrifuge tube, adding 50 µl of RT RNase-free water to the center of the Spin Column and centrifuging for 1 min at 8.000 g (incubation for 2 min before centrifugation!).
C5. The amount of the purified cRNA – assessed by spectrophotometry – was 36,8 µg. The purity (assessed by the OD260/OD280 ratio) was 2,421.

growth_protocol:
The following protocol describes the activation condition of the sample:
For seven days (until total RNA extraction), 1x10^5 purified human naive T cells were co-incubated in a 96 well plate (in 200 µl total volume per well) with 1x10^4 vaccine cells alone. 38 wells were performed in parallel.
Vaccine cells consisted in MCF-7 cells (a mammacarcinoma cell line; # HTB-22, ATCC, Rockville, USA) which were first gamma-irradiated (200 Gy using a 131Cs source; Gammacell 1000, Atomic Energy of Canada) and then modified with Newcastle Disease Virus [NDV; 100 hemagglutination units (HU) per 1x10^7 cells], an avian paramyxovirus which replicates selectively in tumor cells.
This sample served as control sample #4 (effect of vaccine cells alone).

extract_protocol:
Prior RNA extraction, residual contaminating MCF-7 vaccine cells had to be removed from pooled activated naive T cells. To this end, a mouse anti-human epithelial cell adhesion molecule (EpCAM) mAb (clone HEA-125, a kind gift from Dr. G. Moldenhauer, DKFZ, Germany) was first pre-coated onto Pan Mouse IgG Dynabeads (Dynal) according to standard protocol. Briefly, in a microcentrifuge tube 0,5 µg HEA-125 mAb per 1x10^7 Dynabeads was added to the beads. Incubation was carried out for 45 min with gentle tilting and rotation. The tube was placed in an MPC for 1 min, the supernatant discarded, the beads washed twice in 2 ml PBS (containing 1 % FCS), and finally resuspended in a volume of PBS (containing 1 % FCS) to yield a final concentration of 4x10^8 beads per ml.
25 µl of this bead suspension (corresponding to 1x10^7 beads) were added to the naive T cell/vaccine cell mixture (in a 15 ml Falcon tube). The resulting suspension was incubated for 30 min at RT on a rotator and the bead-attached vaccine cells were removed using an MPC. The naive cells were transferred to a fresh 15 ml Falcon tube, centrifuged (8 min at 300 g), resuspended in 1 ml of PBS and transferred to a 1.5 ml RNase-free microcentrifuge tube for subsequent RNA extraction.
Total RNA was extracted using the ArrayGradeTM Total RNA Isolation Kit (GA-013; version 1.0, 3/4/2005), purchased from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. Cell Preparation:
A1. Cells were pelleted in a 1.5 ml RNase-free tube by centrifugation for 5 min at 300 g.
A2. 350 µl of Lysis & Binding Buffer (G6) were added to the pellet.
A3. To reduce viscosity, the lysate was applied to a supplied Filter Column (previously placed into a collection tube) and centrifuged for 1 min at 11.000 g. The Filter Column was discarded.
B. Total RNA Isolation:
B1. To adjust the RNA binding conditions, 350 µl of 70 % ehtonal were added and the sample mixed by vortexing.
B2. The sample was applied to a provided Spin Column (already placed in its own Collection Tube) and centrifuged for 30 sec at 8.000 g. The flow through was discarded (Binding of the RNA to Spin Column).
B3. 350 µl of Desalting Buffer (G15) were added to the Spin Column and centrifuged for 1 min at 11.000 g. The flow through was discarded (Desalting Spin Column).
B4. 200 µl of Pre-wash Buffer (G16) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then 350 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (second wash). Last 200 µl of Washing Buffer working solution were added to the Spin Column and centrifuged for 3 min at 11.000 g to dry the column completely (third wash).
B5. To elute the RNA, the Spin Column was transferred into a fresh 1.5 ml RNase-free microcentrifuge tube, 50 µl of RNase-free water added and centrifuged for 1 min at 11.000 g.
B6. The amount of total RNA – assessed by spectrophotometry – was 3,78 µg. The purity (assessed by the OD260/OD280 ratio) was 1,969.

treatment_protocol:
Peripheral blood mononuclear cells (PBMCs) were isolated via Ficoll-Paque (Amersham Biosciences) centrifugation from fresh heparinized blood of the healthy donor. From total PBMCs, human naive T cells were purified with the T Cell Negative Isolation Kit (Dynal), involving magnetic depletion of non-T cells labeled with mouse monoclonal antibodies (mAbs) against human CD14, CD16a/b, CD56 and HLA-II DR/DP, via Dynabeads coated with human IgG4 against mouse IgG Fc. The addition of a mouse anti-human CD45RO mAb (BD Pharmingen, clone UCHL1) to the above mentioned antibody cocktail ensured the removal of the memory T cell pool, therefore leaving the naive T cells untouched.
The exact protocol of naive T cell isolation from purified PBMCs is given below:
1. Purified PBMCs were brought to a concentration of 1x10^8 cells per ml phosphate buffered saline (PBS).
2. 20 µl of fetal calf serum (FCS) were added per 1x10^7 cells.
3. 20 µl of the T Cell Negative Isolation Kit’s antibody cocktail were added per 1x10^7 cells. The anti-human CD45RO mAb was added at 1 µg per 1x10^7 cells.
4. The suspension was incubated for 20 min at 4 °C with intermittent shaking.
5. Cells were washed once with 1 ml PBS per 1x10^7 cells (centrifugation at 400 g for 8 min) and resuspended in 0.9 ml PBS per 1x10^7 cells.
6. Dynabeads [washed before twice in PBS using the magnetic particle concentrator (MPC)] were added at 4x10^7 beads per 1x10^7 PBMCs in 100 µl (i.e. 4 beads per PBMC in a total volume of 1 ml).
7. The mix was incubated for 30 min at room temperature (RT) on a rotator.
8. The so-formed cell:bead aggregates (corresponding to non-T cells labeled with beads) were removed using the MPC. The supernatant containing the naive T cells were removed, washed again (by centrifugation) and resuspended in 1 to 3 ml of medium.
The purity of the isolated naive human T cells was assessed by double stainings with fluorescent-labeled mouse anti-human mAbs (all purchased from BD Pharmingen) and FACS analysis. The highly pure naive T cells showed the following expression pattern:
CD3+ CD45RA+ CD45RO- CD25- CD107a-. CD3 is a pan-T cell marker, whereas CD45RA and CD45RO are isoforms (splice variants) of the CD45 molecule (the protein tyrosine phosphatase PTRPC) corresponding to naive and memory T cells, respectively. Negativity for the IL-2 receptor alpha chain (CD25) and the lysosomal associated membrane protein-1 (LAMP-1; CD107a) showed that these isolated cells were indeed not activated. CD25 negativity furthermore demonstrated that CD4+CD25+ T regulatory cells were absent from the cell preparation (also confirmed by CD4/CD25 double staining). Finally, CD4 and CD8 stainings revealed that both T cell subpopulations (T helper cells and cytotoxic T cells, respectively) were present within the cell preparation at a ratio of approximately 2:1.

tag: Healthy donor

tag: Female

tag: Caucasian

tag: 30-years old


title: IL-2 alone d7-S6_280705

growth_protocol:
The following protocol describes the activation condition of the sample:
For seven days (until total RNA extraction), 1x10^5 purified human naive T cells were co-incubated in a 96 well plate (in 200 µl total volume per well) with 1x10^4 vaccine cells and one bispecific (bs) single chain (scFv) fusion protein. 38 wells were performed in parallel.
Vaccine cells consisted in MCF-7 cells (a mammacarcinoma cell line; # HTB-22, ATCC, Rockville, USA) which were first gamma-irradiated (200 Gy using a 131Cs source; Gammacell 1000, Atomic Energy of Canada) and then modified with Newcastle Disease Virus [NDV; 100 hemagglutination units (HU) per 1x10^7 cells], an avian paramyxovirus which replicates selectively in tumor cells.
The fusion protein was bsHN-IL-2 alone [used at a concentration of 84,4 units of hemagglutinin-neuraminidase (HN) activity per 1x10^5 naive T cells]. This molecule can attach via its viral HN-specific scFv to the vaccine cells and deliver – via cross-linking – IL-2 (costimulus 2b) signals to the naive T cells.
This sample served as control sample #3 (effect of vaccine cells-attached bsHN-IL-2 alone).

extract_protocol:
Prior RNA extraction, residual contaminating MCF-7 vaccine cells had to be removed from pooled activated naive T cells. To this end, a mouse anti-human epithelial cell adhesion molecule (EpCAM) mAb (clone HEA-125, a kind gift from Dr. G. Moldenhauer, DKFZ, Germany) was first pre-coated onto Pan Mouse IgG Dynabeads (Dynal) according to standard protocol. Briefly, in a microcentrifuge tube 0,5 µg HEA-125 mAb per 1x10^7 Dynabeads was added to the beads. Incubation was carried out for 45 min with gentle tilting and rotation. The tube was placed in an MPC for 1 min, the supernatant discarded, the beads washed twice in 2 ml PBS (containing 1 % FCS), and finally resuspended in a volume of PBS (containing 1 % FCS) to yield a final concentration of 4x10^8 beads per ml.
25 µl of this bead suspension (corresponding to 1x10^7 beads) were added to the naive T cell/vaccine cell mixture (in a 15 ml Falcon tube). The resulting suspension was incubated for 30 min at RT on a rotator and the bead-attached vaccine cells were removed using an MPC. The naive cells were transferred to a fresh 15 ml Falcon tube, centrifuged (8 min at 300 g), resuspended in 1 ml of PBS and transferred to a 1.5 ml RNase-free microcentrifuge tube for subsequent RNA extraction.
Total RNA was extracted using the ArrayGradeTM Total RNA Isolation Kit (GA-013; version 1.0, 3/4/2005), purchased from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. Cell Preparation:
A1. Cells were pelleted in a 1.5 ml RNase-free tube by centrifugation for 5 min at 300 g.
A2. 350 µl of Lysis & Binding Buffer (G6) were added to the pellet.
A3. To reduce viscosity, the lysate was applied to a supplied Filter Column (previously placed into a collection tube) and centrifuged for 1 min at 11.000 g. The Filter Column was discarded.
B. Total RNA Isolation:
B1. To adjust the RNA binding conditions, 350 µl of 70 % ehtonal were added and the sample mixed by vortexing.
B2. The sample was applied to a provided Spin Column (already placed in its own Collection Tube) and centrifuged for 30 sec at 8.000 g. The flow through was discarded (Binding of the RNA to Spin Column).
B3. 350 µl of Desalting Buffer (G15) were added to the Spin Column and centrifuged for 1 min at 11.000 g. The flow through was discarded (Desalting Spin Column).
B4. 200 µl of Pre-wash Buffer (G16) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then 350 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (second wash). Last 200 µl of Washing Buffer working solution were added to the Spin Column and centrifuged for 3 min at 11.000 g to dry the column completely (third wash).
B5. To elute the RNA, the Spin Column was transferred into a fresh 1.5 ml RNase-free microcentrifuge tube, 50 µl of RNase-free water added and centrifuged for 1 min at 11.000 g.
B6. The amount of total RNA – assessed by spectrophotometry – was 4,92 µg. The purity (assessed by the OD260/OD280 ratio) was 2,025.


title: sDf35

source_name: whole animal, mixed stage; Strain: BC2511


title: sDf74

source_name: whole animal, mixed stage; Strain: BC3217

source_name: whole animal, mixed stage; Strain: BC3957


title: sDf40

source_name: whole animal, mixed stage; Strain: BC2738

source_name: whole animal, mixed stage; Strain: BC3470


title: Reference, freshly isolated, purified naive T cells d0-S1_280705

label_protocol:
Total RNA was biotin-labeled using the TrueLabeling-AMPTM Linear RNA Amplification Kit (GA-010; version 1.3, 3/4/2005), purchased from SuperArray Bioscience Corporation.
This kit is designed to linearly amplify and label antisense RNA (aRNA, also known as cRNA: complementary RNA) for hybridization to the Oligo GEArray from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. cDNA synthesis from extracted total RNA:
A1. Preparation of the Annealing Mixture:
1,6 µg of total RNA,
1,0 µl of TrueLabeling Primer (G1),
0,5 µl of CD45 primer (specially designed by SuperArray Bioscience Corporation) and
RNase-free water
were combined in a sterile PCR tube for a final volume of 10 µl.
The CD45 primer was needed in the reaction to facilitate target labeling for alternative spliced CD45 transcripts. Because of lack of appropriate testing material for alternative CD45 transcripts, SuperArray Bioscience Corporation was unable to confirm the effectiveness of this primer in their QC experiment. Therefore, no guaranty was given for the performance of this primer.
A2. Preparation of cDNA Synthesis Reaction:
To the 10 µl annealing mixture, the following components were added (final volume: 20 µl):
4 µl RNase-free water,
4 µl 5 X cDNA Synthesis Buffer (G3),
1 µl RNase Inhibitor (RI) and
cDNA Synthesis Enzyme Mix (G2).
The reaction was carried out by incubation for 50 minutes at 42 °C.
B. cRNA Synthesis, Labeling and Amplification from cDNA:
To the 20 µl 37 °C-warm cDNA synthesis reaction, the following components were added in the following order (final volume: 40 µl)
16 µl 2,5 X RNA Amplification Buffer (G4),
2 µl Biotin-16-uridine-5’-triphosphate (Roche, # 11 388 908 910) and
2 µl Amplification Enzyme Mix (G5).
The reaction was carried out by incubation overnight (18 hours) at 37 °C.
C. cRNA Purification:
The purification of the labeled and amplified cRNA was performed using the ArrayGrade cRNA Cleanup Kit (GA-012; version 1.3, 3/4/2005).
C1. The cRNA sample was prepared by (i) adding on ice 60 µl of RNase-free water for a final volume of 100 µl (the reaction was then transferred to a 1.5 ml RNase-free microcentrifuge tube), (ii) adding 350 µl Lysis & Binding Buffer (G6) and (iii) immediately adding 350 µl of RT 100 % ethanol.
C2. The cRNA was loaded onto a supplied Spin Column and centrifuged for 30 sec at 8.000 g (binding of the cRNA to the Spin Column). The flow through was discarded.
C3. 600 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then another 200 µl of Washing Buffer working solution were applied and centrifuged for 3 min at 11.000 g (second wash).
C4. cRNA was eluted from the Spin Column by placing the Spin Column onto a fresh 1.5 ml RNase-free microcentrifuge tube, adding 50 µl of RT RNase-free water to the center of the Spin Column and centrifuging for 1 min at 8.000 g (incubation for 2 min before centrifugation!).
C5. The amount of the purified cRNA – assessed by spectrophotometry – was 46,2 µg. The purity (assessed by the OD260/OD280 ratio) was 2,287.

growth_protocol: 1x10^7 freshly purified naive T cells were directly used for total RNA extraction (i.e. ex vivo, without previous activation). This sample was used as reference.

extract_protocol:
Total RNA was extracted using the ArrayGradeTM Total RNA Isolation Kit (GA-013; version 1.0, 3/4/2005), purchased from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. Cell Preparation:
A1. Cells were pelleted in a 1.5 ml RNase-free tube by centrifugation for 5 min at 300 g.
A2. 350 µl of Lysis & Binding Buffer (G6) were added to the pellet.
A3. To reduce viscosity, the lysate was applied to a supplied Filter Column (previously placed into a collection tube) and centrifuged for 1 min at 11.000 g. The Filter Column was discarded.
B. Total RNA Isolation:
B1. To adjust the RNA binding conditions, 350 µl of 70 % ethanol were added and the sample mixed by vortexing.
B2. The sample was applied to a provided Spin Column (already placed in its own collection tube) and centrifuged for 30 sec at 8.000 g. The flow through was discarded (Binding of the RNA to Spin Column).
B3. 350 µl of Desalting Buffer (G15) were added to the Spin Column and centrifuged for 1 min at 11.000 g. The flow through was discarded (Desalting Spin Column).
B4. 200 µl of Pre-wash Buffer (G16) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then 350 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (second wash). Last, 200 µl of Washing Buffer working solution were added to the Spin Column and centrifuged for 3 min at 11.000 g to dry the column completely (third wash).
B5. To elute the RNA, the Spin Column was transferred into a fresh 1.5 ml RNase-free microcentrifuge tube, 50 µl of RNase-free water added and centrifuged for 1 min at 11.000 g.
B6. The amount of total RNA – as assessed by spectrophotometry – was 7,60 µg. The purity (assessed by the OD260/OD280 ratio) was 1,900.

treatment_protocol:
Peripheral blood mononuclear cells (PBMCs) were isolated via Ficoll-Paque (Amersham Biosciences) centrifugation from fresh heparinized blood of the healthy donor. From total PBMCs, human naive T cells were purified with the T Cell Negative Isolation Kit (Dynal), involving magnetic depletion of non-T cells labeled with mouse monoclonal antibodies (mAbs) against human CD14, CD16a/b, CD56 and HLA-II DR/DP, via Dynabeads coated with human IgG4 against mouse IgG Fc. The addition of a mouse anti-human CD45RO mAb (BD Pharmingen, clone UCHL1) to the above mentioned antibody cocktail ensured the removal of the memory T cell pool, therefore leaving the naive T cells untouched.
The exact protocol of naive T cell isolation from purified PBMCs is given below:
1. Purified PBMCs were brought to a concentration of 1x10^8 cells per ml phosphate buffered saline (PBS).
2. 20 µl of fetal calf serum (FCS) were added per 1x10^7 cells.
3. 20 µl of the T Cell Negative Isolation Kit's antibody cocktail were added per 1x10^7 cells. The anti-human CD45RO mAb was added at 1 µg per 1x10^7 cells.
4. The suspension was incubated for 20 min at 4 °C with intermittent shaking.
5. Cells were washed once with 1 ml PBS per 1x10^7 cells (centrifugation at 400 g for 8 min) and resuspended in 0.9 ml PBS per 1x10^7 cells.
6. Dynabeads [washed before twice in PBS using the magnetic particle concentrator (MPC)] were added at 4x10^7 beads per 1x10^7 PBMCs in 100 µl (i.e. 4 beads per PBMC in a total volume of 1 ml).
7. The mix was incubated for 30 min at room temperature (RT) on a rotator.
8. The so-formed cell:bead aggregates (corresponding to non-T cells labeled with beads) were removed using the MPC. The supernatant containing the naive T cells were removed, washed again (by centrifugation) and resuspended in 1 to 3 ml of medium.
The purity of the isolated naive human T cells was assessed by double stainings with fluorescent-labeled mouse anti-human mAbs (all purchased from BD Pharmingen) and FACS analysis. The highly pure naive T cells showed the following expression pattern:
CD3+ CD45RA+ CD45RO- CD25- CD107a-. CD3 is a pan-T cell marker, whereas CD45RA and CD45RO are isoforms (splice variants) of the CD45 molecule (the protein tyrosine phosphatase PTRPC) corresponding to naive and memory T cells, respectively. Negativity for the IL-2 receptor alpha chain (CD25) and the lysosomal associated membrane protein-1 (LAMP-1; CD107a) showed that these isolated cells were indeed not activated. CD25 negativity furthermore demonstrated that CD4+CD25+ T regulatory cells were absent from the cell preparation (also confirmed by CD4/CD25 double staining). Finally, CD4 and CD8 stainings revealed that both T cell subpopulations (T helper cells and cytotoxic T cells, respectively) were present within the cell preparation at a ratio of approximately 2:1.


title: anti-CD3/anti-CD28 activation d7-S2_280705

label_protocol:
Total RNA was biotin-labeled using the TrueLabeling-AMPTM Linear RNA Amplification Kit (GA-010; version 1.3, 3/4/2005), purchased from SuperArray Bioscience Corporation.
This kit is designed to linearly amplify and label antisense RNA (aRNA, also known as cRNA: complementary RNA) for hybridization to the Oligo GEArray from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. cDNA synthesis from extracted total RNA:
A1. Preparation of the Annealing Mixture:
2,0 µg of total RNA,
1,0 µl of TrueLabeling Primer (G1),
0,5 µl of CD45 primer (specially designed by SuperArray Bioscience Corporation) and
RNase-free water
were combined in a sterile PCR tube for a final volume of 10 µl.
The CD45 primer was needed in the reaction to facilitate target labeling for alternative spliced CD45 transcripts. Because of lack of appropriate testing material for alternative CD45 transcripts, SuperArray Bioscience Corporation was unable to confirm the effectiveness of this primer in their QC experiment. Therefore, no guaranty was given for the performance of this primer.
A2. Preparation of cDNA Synthesis Reaction:
To the 10 µl annealing mixture, the following components were added (final volume: 20 µl):
4 µl RNase-free water,
4 µl 5 X cDNA Synthesis Buffer (G3),
1 µl RNase Inhibitor (RI) and
cDNA Synthesis Enzyme Mix (G2).
The reaction was carried out by incubation for 50 minutes at 42 °C.
B. cRNA Synthesis, Labeling and Amplification from cDNA:
To the 20 µl 37 °C-warm cDNA synthesis reaction, the following components were added in the following order (final volume: 40 µl)
16 µl 2,5 X RNA Amplification Buffer (G4),
2 µl Biotin-16-uridine-5’-triphosphate (Roche, # 11 388 908 910) and
2 µl Amplification Enzyme Mix (G5).
The reaction was carried out by incubation overnight (18 hours) at 37 °C.
C. cRNA Purification:
The purification of the labeled and amplified cRNA was performed using the ArrayGrade cRNA Cleanup Kit (GA-012; version 1.3, 3/4/2005).
C1. The cRNA sample was prepared by (i) adding on ice 60 µl of RNase-free water for a final volume of 100 µl (the reaction was then transferred to a 1.5 ml RNase-free microcentrifuge tube), (ii) adding 350 µl Lysis & Binding Buffer (G6) and (iii) immediately adding 350 µl of RT 100 % ethanol.
C2. The cRNA was loaded onto a supplied Spin Column and centrifuged for 30 sec at 8.000 g (binding of the cRNA to the Spin Column). The flow through was discarded.
C3. 600 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then another 200 µl of Washing Buffer working solution were applied and centrifuged for 3 min at 11.000 g (second wash).
C4. cRNA was eluted from the Spin Column by placing the Spin Column onto a fresh 1.5 ml RNase-free microcentrifuge tube, adding 50 µl of RT RNase-free water to the center of the Spin Column and centrifuging for 1 min at 8.000 g (incubation for 2 min before centrifugation!).
C5. The amount of the purified cRNA – assessed by spectrophotometry – was 35,6 µg. The purity (assessed by the OD260/OD280 ratio) was 2,472.

growth_protocol:
The following protocol describes the activation condition of the sample:
For seven days (until total RNA extraction), 1x10^5 purified human naive T cells were co-incubated in a 96 well plate (in 200 µl total volume per well) with 1x10^4 vaccine cells and bispecific (bs) single chain (scFv) fusion proteins. 18 wells were performed in parallel.
Vaccine cells consisted in MCF-7 cells (a mammacarcinoma cell line; # HTB-22, ATCC, Rockville, USA) which were first gamma-irradiated (200 Gy using a 131Cs source; Gammacell 1000, Atomic Energy of Canada) and then modified with Newcastle Disease Virus [NDV; 100 hemagglutination units (HU) per 1x10^7 cells], an avian paramyxovirus which replicates selectively in tumor cells.
The fusion proteins were bsHN-CD3 (used at a suboptimal concentration of 500 pg per 1x10^5 naive T cells) and bsHN-CD28 [used at a concentration of 84,4 units of hemagglutinin-neuraminidase (HN) activity per 1x10^5 naive T cells]. These molecules can attach via their viral HN-specific scFv to the vaccine cells and deliver – via cross-linking – anti-CD3 (signal 1) and anti-CD28 (costimulus 2a) signals to the naive T cells.
This sample was test sample #1 (effect of costimulatory capacity of bsHN-CD28 in the presence of a suboptimal dose of bsHN-CD3).

extract_protocol:
Prior RNA extraction, residual contaminating MCF-7 vaccine cells had to be removed from pooled activated naive T cells. To this end, a mouse anti-human epithelial cell adhesion molecule (EpCAM) mAb (clone HEA-125, a kind gift from Dr. G. Moldenhauer, DKFZ, Germany) was first pre-coated onto Pan Mouse IgG Dynabeads (Dynal) according to standard protocol. Briefly, in a microcentrifuge tube 0,5 µg HEA-125 mAb per 1x10^7 Dynabeads was added to the beads. Incubation was carried out for 45 min with gentle tilting and rotation. The tube was placed in an MPC for 1 min, the supernatant discarded, the beads washed twice in 2 ml PBS (containing 1 % FCS), and finally resuspended in a volume of PBS (containing 1 % FCS) to yield a final concentration of 4x10^8 beads per ml.
25 µl of this bead suspension (corresponding to 1x10^7 beads) were added to the naive T cell/vaccine cell mixture (in a 15 ml Falcon tube). The resulting suspension was incubated for 30 min at RT on a rotator and the bead-attached vaccine cells were removed using an MPC. The naive cells were transferred to a fresh 15 ml Falcon tube, centrifuged (8 min at 300 g), resuspended in 1 ml of PBS and transferred to a 1.5 ml RNase-free microcentrifuge tube for subsequent RNA extraction.
Total RNA was extracted using the ArrayGradeTM Total RNA Isolation Kit (GA-013; version 1.0, 3/4/2005), purchased from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. Cell Preparation:
A1. Cells were pelleted in a 1.5 ml RNase-free tube by centrifugation for 5 min at 300 g.
A2. 350 µl of Lysis & Binding Buffer (G6) were added to the pellet.
A3. To reduce viscosity, the lysate was applied to a supplied Filter Column (previously placed into a collection tube) and centrifuged for 1 min at 11.000 g. The Filter Column was discarded.
B. Total RNA Isolation:
B1. To adjust the RNA binding conditions, 350 µl of 70 % ehtonal were added and the sample mixed by vortexing.
B2. The sample was applied to a provided Spin Column (already placed in its own Collection Tube) and centrifuged for 30 sec at 8.000 g. The flow through was discarded (Binding of the RNA to Spin Column).
B3. 350 µl of Desalting Buffer (G15) were added to the Spin Column and centrifuged for 1 min at 11.000 g. The flow through was discarded (Desalting Spin Column).
B4. 200 µl of Pre-wash Buffer (G16) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then 350 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (second wash). Last 200 µl of Washing Buffer working solution were added to the Spin Column and centrifuged for 3 min at 11.000 g to dry the column completely (third wash).
B5. To elute the RNA, the Spin Column was transferred into a fresh 1.5 ml RNase-free microcentrifuge tube, 50 µl of RNase-free water added and centrifuged for 1 min at 11.000 g.
B6. The amount of total RNA – assessed by spectrophotometry – was 12,96 µg. The purity (assessed by the OD260/OD280 ratio) was 1,800.


title: anti-CD28 alone d7-S5_280705

label_protocol:
Total RNA was biotin-labeled using the TrueLabeling-AMPTM Linear RNA Amplification Kit (GA-010; version 1.3, 3/4/2005), purchased from SuperArray Bioscience Corporation.
This kit is designed to linearly amplify and label antisense RNA (aRNA, also known as cRNA: complementary RNA) for hybridization to the Oligo GEArray from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. cDNA synthesis from extracted total RNA:
A1. Preparation of the Annealing Mixture:
1,6 µg of total RNA,
1,0 µl of TrueLabeling Primer (G1),
0,5 µl of CD45 primer (specially designed by SuperArray Bioscience Corporation) and
RNase-free water
were combined in a sterile PCR tube  for a final volume of 10 µl.
The CD45 primer was needed in the reaction to facilitate target labeling for alternative spliced CD45 transcripts. Because of lack of appropriate testing material for alternative CD45 transcripts, SuperArray Bioscience Corporation was unable to confirm the effectiveness of this primer in their QC experiment. Therefore, no guaranty was given for the performance of this primer.
A2. Preparation of cDNA Synthesis Reaction:
To the 10 µl annealing mixture, the following components were added (final volume: 20 µl):
4 µl RNase-free water,
4 µl 5 X cDNA Synthesis Buffer (G3),
1 µl RNase Inhibitor (RI) and
cDNA Synthesis Enzyme Mix (G2).
The reaction was carried out by incubation for 50 minutes at 42 °C.
B. cRNA Synthesis, Labeling and Amplification from cDNA:
To the 20 µl 37 °C-warm cDNA synthesis reaction, the following components were added in the following order (final volume: 40 µl)
16 µl 2,5 X RNA Amplification Buffer (G4),
2 µl Biotin-16-uridine-5’-triphosphate (Roche, # 11 388 908 910) and
2 µl Amplification Enzyme Mix (G5).
The reaction was carried out by incubation overnight (18 hours) at 37 °C.
C. cRNA Purification:
The purification of the labeled and amplified cRNA was performed using the ArrayGrade cRNA Cleanup Kit (GA-012; version 1.3, 3/4/2005).
C1. The cRNA sample was prepared by (i) adding on ice 60 µl of RNase-free water for a final volume of 100 µl (the reaction was then transferred to a 1.5 ml RNase-free microcentrifuge tube), (ii) adding 350 µl Lysis & Binding Buffer (G6) and (iii) immediately adding 350 µl of RT 100 % ethanol.
C2. The cRNA was loaded onto a supplied Spin Column and centrifuged for 30 sec at 8.000 g (binding of the cRNA to the Spin Column). The flow through was discarded.
C3. 600 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then another 200 µl of Washing Buffer working solution were applied and centrifuged for 3 min at 11.000 g (second wash).
C4. cRNA was eluted from the Spin Column by placing the Spin Column onto a fresh 1.5 ml RNase-free microcentrifuge tube, adding 50 µl of RT RNase-free water to the center of the Spin Column and centrifuging for 1 min at 8.000 g (incubation for 2 min before centrifugation!).
C5. The amount of the purified cRNA – assessed by spectrophotometry – was 46,0 µg. The purity (assessed by the OD260/OD280 ratio) was 2,473.

growth_protocol:
The following protocol describes the activation condition of the sample:
For seven days (until total RNA extraction), 1x10^5 purified human naive T cells were co-incubated in a 96 well plate (in 200 µl total volume per well) with 1x10^4 vaccine cells and one bispecific (bs) single chain (scFv) fusion protein. 38 wells were performed in parallel.
Vaccine cells consisted in MCF-7 cells (a mammacarcinoma cell line; # HTB-22, ATCC, Rockville, USA) which were first gamma-irradiated (200 Gy using a 131Cs source; Gammacell 1000, Atomic Energy of Canada) and then modified with Newcastle Disease Virus [NDV; 100 hemagglutination units (HU) per 1x10^7 cells], an avian paramyxovirus which replicates selectively in tumor cells.
The fusion protein was bsHN-CD28 alone [used at a concentration of 84,4 units of hemagglutinin-neuraminidase (HN) activity per 1x10^5 naive T cells]. This molecule can attach via its viral HN-specific scFv to the vaccine cells and deliver – via cross-linking – anti-CD28 (costimulus 2a) signals to the naive T cells.
This sample served as control sample #2 (effect of vaccine cells-attached bsHN-CD28 alone).

extract_protocol:
Prior RNA extraction, residual contaminating MCF-7 vaccine cells had to be removed from pooled activated naive T cells. To this end, a mouse anti-human epithelial cell adhesion molecule (EpCAM) mAb (clone HEA-125, a kind gift from Dr. G. Moldenhauer, DKFZ, Germany) was first pre-coated onto Pan Mouse IgG Dynabeads (Dynal) according to standard protocol. Briefly, in a microcentrifuge tube 0,5 µg HEA-125 mAb per 1x10^7 Dynabeads was added to the beads. Incubation was carried out for 45 min with gentle tilting and rotation. The tube was placed in an MPC for 1 min, the supernatant discarded, the beads washed twice in 2 ml PBS (containing 1 % FCS), and finally resuspended in a volume of PBS (containing 1 % FCS) to yield a final concentration of 4x10^8 beads per ml.
25 µl of this bead suspension (corresponding to 1x10^7 beads) were added to the naive T cell/vaccine cell mixture (in a 15 ml Falcon tube). The resulting suspension was incubated for 30 min at RT on a rotator and the bead-attached vaccine cells were removed using an MPC. The naive cells were transferred to a fresh 15 ml Falcon tube, centrifuged (8 min at 300 g), resuspended in 1 ml of PBS and transferred to a 1.5 ml RNase-free microcentrifuge tube for subsequent RNA extraction.
Total RNA was extracted using the ArrayGradeTM Total RNA Isolation Kit (GA-013; version 1.0, 3/4/2005), purchased from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. Cell Preparation:
A1. Cells were pelleted in a 1.5 ml RNase-free tube by centrifugation for 5 min at 300 g.
A2. 350 µl of Lysis & Binding Buffer (G6) were added to the pellet.
A3. To reduce viscosity, the lysate was applied to a supplied Filter Column (previously placed into a collection tube) and centrifuged for 1 min at 11.000 g. The Filter Column was discarded.
B. Total RNA Isolation:
B1. To adjust the RNA binding conditions, 350 µl of 70 % ehtonal were added and the sample mixed by vortexing.
B2. The sample was applied to a provided Spin Column (already placed in its own Collection Tube) and centrifuged for 30 sec at 8.000 g. The flow through was discarded (Binding of the RNA to Spin Column).
B3. 350 µl of Desalting Buffer (G15) were added to the Spin Column and centrifuged for 1 min at 11.000 g. The flow through was discarded (Desalting Spin Column).
B4. 200 µl of Pre-wash Buffer (G16) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then 350 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (second wash). Last 200 µl of Washing Buffer working solution were added to the Spin Column and centrifuged for 3 min at 11.000 g to dry the column completely (third wash).
B5. To elute the RNA, the Spin Column was transferred into a fresh 1.5 ml RNase-free microcentrifuge tube, 50 µl of RNase-free water added and centrifuged for 1 min at 11.000 g.
B6. The amount of total RNA – assessed by spectrophotometry – was 2,76 µg. The purity (assessed by the OD260/OD280 ratio) was 2,000.


title: anti-CD3 alone d7-S4_280705

label_protocol:
Total RNA was biotin-labeled using the TrueLabeling-AMPTM Linear RNA Amplification Kit (GA-010; version 1.3, 3/4/2005), purchased from SuperArray Bioscience Corporation.
This kit is designed to linearly amplify and label antisense RNA (aRNA, also known as cRNA: complementary RNA) for hybridization to the Oligo GEArray from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. cDNA synthesis from extracted total RNA:
A1. Preparation of the Annealing Mixture:
2,0 µg of total RNA,
1,0 µl of TrueLabeling Primer (G1),
0,5 µl of CD45 primer (specially designed by SuperArray Bioscience Corporation) and
RNase-free water
were combined in a sterile PCR tube for a final volume of 10 µl.
The CD45 primer was needed in the reaction to facilitate target labeling for alternative spliced CD45 transcripts. Because of lack of appropriate testing material for alternative CD45 transcripts, SuperArray Bioscience Corporation was unable to confirm the effectiveness of this primer in their QC experiment. Therefore, no guaranty was given for the performance of this primer.
A2. Preparation of cDNA Synthesis Reaction:
To the 10 µl annealing mixture, the following components were added (final volume: 20 µl):
4 µl RNase-free water,
4 µl 5 X cDNA Synthesis Buffer (G3),
1 µl RNase Inhibitor (RI) and
cDNA Synthesis Enzyme Mix (G2).
The reaction was carried out by incubation for 50 minutes at 42 °C.
B. cRNA Synthesis, Labeling and Amplification from cDNA:
To the 20 µl 37 °C-warm cDNA synthesis reaction, the following components were added in the following order (final volume: 40 µl)
16 µl 2,5 X RNA Amplification Buffer (G4),
2 µl Biotin-16-uridine-5’-triphosphate (Roche, # 11 388 908 910) and
2 µl Amplification Enzyme Mix (G5).
The reaction was carried out by incubation overnight (18 hours) at 37 °C.
C. cRNA Purification:
The purification of the labeled and amplified cRNA was performed using the ArrayGrade cRNA Cleanup Kit (GA-012; version 1.3, 3/4/2005).
C1. The cRNA sample was prepared by (i) adding on ice 60 µl of RNase-free water for a final volume of 100 µl (the reaction was then transferred to a 1.5 ml RNase-free microcentrifuge tube), (ii) adding 350 µl Lysis & Binding Buffer (G6) and (iii) immediately adding 350 µl of RT 100 % ethanol.
C2. The cRNA was loaded onto a supplied Spin Column and centrifuged for 30 sec at 8.000 g (binding of the cRNA to the Spin Column). The flow through was discarded.
C3. 600 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then another 200 µl of Washing Buffer working solution were applied and centrifuged for 3 min at 11.000 g (second wash).
C4. cRNA was eluted from the Spin Column by placing the Spin Column onto a fresh 1.5 ml RNase-free microcentrifuge tube, adding 50 µl of RT RNase-free water to the center of the Spin Column and centrifuging for 1 min at 8.000 g (incubation for 2 min before centrifugation!).
C5. The amount of the purified cRNA – assessed by spectrophotometry – was 45,2 µg. The purity (assessed by the OD260/OD280 ratio) was 2,457.

growth_protocol:
The following protocol describes the activation condition of the sample:
For seven days (until total RNA extraction), 1x10^5 purified human naive T cells were co-incubated in a 96 well plate (in 200 µl total volume per well) with 1x10^4 vaccine cells and one bispecific (bs) single chain (scFv) fusion protein. 38 wells were performed in parallel.
Vaccine cells consisted in MCF-7 cells (a mammacarcinoma cell line; # HTB-22, ATCC, Rockville, USA) which were first gamma-irradiated (200 Gy using a 131Cs source; Gammacell 1000, Atomic Energy of Canada) and then modified with Newcastle Disease Virus [NDV; 100 hemagglutination units (HU) per 1x10^7 cells], an avian paramyxovirus which replicates selectively in tumor cells.
The fusion protein was bsHN-CD3 alone (used at a suboptimal concentration of 500 pg per 1x10^5 naive T cells). This molecule can attach via its viral HN-specific scFv to the vaccine cells and deliver – via cross-linking – anti-CD3 (signal 1) signals to the naive T cells.
This sample served as control sample #1 (effect of vaccine cells-attached bsHN-CD3 alone).

extract_protocol:
Prior RNA extraction, residual contaminating MCF-7 vaccine cells had to be removed from pooled activated naive T cells. To this end, a mouse anti-human epithelial cell adhesion molecule (EpCAM) mAb (clone HEA-125, a kind gift from Dr. G. Moldenhauer, DKFZ, Germany) was first pre-coated onto Pan Mouse IgG Dynabeads (Dynal) according to standard protocol. Briefly, in a microcentrifuge tube 0,5 µg HEA-125 mAb per 1x10^7 Dynabeads was added to the beads. Incubation was carried out for 45 min with gentle tilting and rotation. The tube was placed in an MPC for 1 min, the supernatant discarded, the beads washed twice in 2 ml PBS (containing 1 % FCS), and finally resuspended in a volume of PBS (containing 1 % FCS) to yield a final concentration of 4x10^8 beads per ml.
25 µl of this bead suspension (corresponding to 1x10^7 beads) were added to the naive T cell/vaccine cell mixture (in a 15 ml Falcon tube). The resulting suspension was incubated for 30 min at RT on a rotator and the bead-attached vaccine cells were removed using an MPC. The naive cells were transferred to a fresh 15 ml Falcon tube, centrifuged (8 min at 300 g), resuspended in 1 ml of PBS and transferred to a 1.5 ml RNase-free microcentrifuge tube for subsequent RNA extraction.
Total RNA was extracted using the ArrayGradeTM Total RNA Isolation Kit (GA-013; version 1.0, 3/4/2005), purchased from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. Cell Preparation:
A1. Cells were pelleted in a 1.5 ml RNase-free tube by centrifugation for 5 min at 300 g.
A2. 350 µl of Lysis & Binding Buffer (G6) were added to the pellet.
A3. To reduce viscosity, the lysate was applied to a supplied Filter Column (previously placed into a collection tube) and centrifuged for 1 min at 11.000 g. The Filter Column was discarded.
B. Total RNA Isolation:
B1. To adjust the RNA binding conditions, 350 µl of 70 % ehtonal were added and the sample mixed by vortexing.
B2. The sample was applied to a provided Spin Column (already placed in its own Collection Tube) and centrifuged for 30 sec at 8.000 g. The flow through was discarded (Binding of the RNA to Spin Column).
B3. 350 µl of Desalting Buffer (G15) were added to the Spin Column and centrifuged for 1 min at 11.000 g. The flow through was discarded (Desalting Spin Column).
B4. 200 µl of Pre-wash Buffer (G16) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then 350 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (second wash). Last 200 µl of Washing Buffer working solution were added to the Spin Column and centrifuged for 3 min at 11.000 g to dry the column completely (third wash).
B5. To elute the RNA, the Spin Column was transferred into a fresh 1.5 ml RNase-free microcentrifuge tube, 50 µl of RNase-free water added and centrifuged for 1 min at 11.000 g.
B6. The amount of total RNA – assessed by spectrophotometry – was 3,96 µg. The purity (assessed by the OD260/OD280 ratio) was 1,985.


title: sDf27

source_name: whole animal, mixed stage; Strain: BC1230


title: anti-CD3/IL-2 activation d7-S3_280705

label_protocol:
Total RNA was biotin-labeled using the TrueLabeling-AMPTM Linear RNA Amplification Kit (GA-010; version 1.3, 3/4/2005), purchased from SuperArray Bioscience Corporation.
This kit is designed to linearly amplify and label antisense RNA (aRNA, also known as cRNA: complementary RNA) for hybridization to the Oligo GEArray from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. cDNA synthesis from extracted total RNA:
A1. Preparation of the Annealing Mixture:
2,0 µg of total RNA,
1,0 µl of TrueLabeling Primer (G1),
0,5 µl of CD45 primer (specially designed by SuperArray Bioscience Corporation) and
RNase-free water
were combined in a sterile PCR tube for a final volume of 10 µl.
The CD45 primer was needed in the reaction to facilitate target labeling for alternative spliced CD45 transcripts. Because of lack of appropriate testing material for alternative CD45 transcripts, SuperArray Bioscience Corporation was unable to confirm the effectiveness of this primer in their QC experiment. Therefore, no guaranty was given for the performance of this primer.
A2. Preparation of cDNA Synthesis Reaction:
To the 10 µl annealing mixture, the following components were added (final volume: 20 µl):
4 µl RNase-free water,
4 µl 5 X cDNA Synthesis Buffer (G3),
1 µl RNase Inhibitor (RI) and
cDNA Synthesis Enzyme Mix (G2).
The reaction was carried out by incubation for 50 minutes at 42 °C.
B. cRNA Synthesis, Labeling and Amplification from cDNA:
To the 20 µl 37 °C-warm cDNA synthesis reaction, the following components were added in the following order (final volume: 40 µl)
16 µl 2,5 X RNA Amplification Buffer (G4),
2 µl Biotin-16-uridine-5’-triphosphate (Roche, # 11 388 908 910) and
2 µl Amplification Enzyme Mix (G5).
The reaction was carried out by incubation overnight (18 hours) at 37 °C.
C. cRNA Purification:
The purification of the labeled and amplified cRNA was performed using the ArrayGrade cRNA Cleanup Kit (GA-012; version 1.3, 3/4/2005).
C1. The cRNA sample was prepared by (i) adding on ice 60 µl of RNase-free water for a final volume of 100 µl (the reaction was then transferred to a 1.5 ml RNase-free microcentrifuge tube), (ii) adding 350 µl Lysis & Binding Buffer (G6) and (iii) immediately adding 350 µl of RT 100 % ethanol.
C2. The cRNA was loaded onto a supplied Spin Column and centrifuged for 30 sec at 8.000 g (binding of the cRNA to the Spin Column). The flow through was discarded.
C3. 600 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then another 200 µl of Washing Buffer working solution were applied and centrifuged for 3 min at 11.000 g (second wash).
C4. cRNA was eluted from the Spin Column by placing the Spin Column onto a fresh 1.5 ml RNase-free microcentrifuge tube, adding 50 µl of RT RNase-free water to the center of the Spin Column and centrifuging for 1 min at 8.000 g (incubation for 2 min before centrifugation!).
C5. The amount of the purified cRNA – assessed by spectrophotometry – was 33,8 µg. The purity (assessed by the OD260/OD280 ratio) was 2,449.

growth_protocol:
The following protocol describes the activation condition of the sample:
For seven days (until total RNA extraction), 1x10^5 purified human naive T cells were co-incubated in a 96 well plate (in 200 µl total volume per well) with 1x10^4 vaccine cells and bispecific (bs) single chain (scFv) fusion proteins. 18 wells were performed in parallel.
Vaccine cells consisted in MCF-7 cells (a mammacarcinoma cell line; # HTB-22, ATCC, Rockville, USA) which were first gamma-irradiated (200 Gy using a 131Cs source; Gammacell 1000, Atomic Energy of Canada) and then modified with Newcastle Disease Virus [NDV; 100 hemagglutination units (HU) per 1x10^7 cells], an avian paramyxovirus which replicates selectively in tumor cells.
The fusion proteins were bsHN-CD3 (used at a suboptimal concentration of 500 pg per 1x10^5 naive T cells) and bsHN-IL-2 [used at a concentration of 84,4 units of hemagglutinin-neuraminidase (HN) activity per 1x10^5 naive T cells]. These molecules can attach via their viral HN-specific scFv to the vaccine cells and deliver – via cross-linking – anti-CD3 (signal 1) and IL-2 (costimulus 2b) signals to the naive T cells.
This sample was test sample #2 (effect of costimulatory capacity of bsHN-IL-2 in the presence of a suboptimal dose of bsHN-CD3).

extract_protocol:
Prior RNA extraction, residual contaminating MCF-7 vaccine cells had to be removed from pooled activated naive T cells. To this end, a mouse anti-human epithelial cell adhesion molecule (EpCAM) mAb (clone HEA-125, a kind gift from Dr. G. Moldenhauer, DKFZ, Germany) was first pre-coated onto Pan Mouse IgG Dynabeads (Dynal) according to standard protocol. Briefly, in a microcentrifuge tube 0,5 µg HEA-125 mAb per 1x10^7 Dynabeads was added to the beads. Incubation was carried out for 45 min with gentle tilting and rotation. The tube was placed in an MPC for 1 min, the supernatant discarded, the beads washed twice in 2 ml PBS (containing 1 % FCS), and finally resuspended in a volume of PBS (containing 1 % FCS) to yield a final concentration of 4x10^8 beads per ml.
25 µl of this bead suspension (corresponding to 1x10^7 beads) were added to the naive T cell/vaccine cell mixture (in a 15 ml Falcon tube). The resulting suspension was incubated for 30 min at RT on a rotator and the bead-attached vaccine cells were removed using an MPC. The naive cells were transferred to a fresh 15 ml Falcon tube, centrifuged (8 min at 300 g), resuspended in 1 ml of PBS and transferred to a 1.5 ml RNase-free microcentrifuge tube for subsequent RNA extraction.
Total RNA was extracted using the ArrayGradeTM Total RNA Isolation Kit (GA-013; version 1.0, 3/4/2005), purchased from SuperArray Bioscience Corporation.
The detailed protocol is given:
A. Cell Preparation:
A1. Cells were pelleted in a 1.5 ml RNase-free tube by centrifugation for 5 min at 300 g.
A2. 350 µl of Lysis & Binding Buffer (G6) were added to the pellet.
A3. To reduce viscosity, the lysate was applied to a supplied Filter Column (previously placed into a collection tube) and centrifuged for 1 min at 11.000 g. The Filter Column was discarded.
B. Total RNA Isolation:
B1. To adjust the RNA binding conditions, 350 µl of 70 % ehtonal were added and the sample mixed by vortexing.
B2. The sample was applied to a provided Spin Column (already placed in its own Collection Tube) and centrifuged for 30 sec at 8.000 g. The flow through was discarded (Binding of the RNA to Spin Column).
B3. 350 µl of Desalting Buffer (G15) were added to the Spin Column and centrifuged for 1 min at 11.000 g. The flow through was discarded (Desalting Spin Column).
B4. 200 µl of Pre-wash Buffer (G16) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (first wash). Then 350 µl of Washing Buffer working solution (working solution was prepared by adding 4 ml of 100 % ethanol per 1 ml of Washing Buffer G17) were added to the Spin Column and centrifuged for 30 sec at 8.000 g. The flow through was discarded (second wash). Last 200 µl of Washing Buffer working solution were added to the Spin Column and centrifuged for 3 min at 11.000 g to dry the column completely (third wash).
B5. To elute the RNA, the Spin Column was transferred into a fresh 1.5 ml RNase-free microcentrifuge tube, 50 µl of RNase-free water added and centrifuged for 1 min at 11.000 g.
B6. The amount of total RNA – assessed by spectrophotometry – was 13,20 µg. The purity (assessed by the OD260/OD280 ratio) was 1,833.


title: sDf50

source_name: whole animal, mixed stage; Strain: BC3539


title: sDf33

source_name: whole animal, mixed stage; Strain: BC1781


title: nDf18

source_name: whole animal, mixed stage; Strain: BC1999

source_name: whole animal, mixed stage; Strain: BC2842


title: sDf48

source_name: whole animal, mixed stage; Strain: BC3033


title: Ezh2cKO E16 basal cells, sample 1

city: New York

first: Elena

last: Ezhkova

address: 1428 Madison Avenue, Atran Building, AB7-10B

department: DRB

institute: MSSM

description: Replicate 1 of 2.

hyb_protocol: Performed by the Genomics Core Facility of MSKCC.

scan_protocol: Arrays were scanned per the manufacturer's specifications for the Affymetrix MOE430v2 chip.

source_name: Skin, Ezh2cKO

extract_protocol: Stratagene Absolutely RNA kit

tag: Mice

value: Mixed strain (C57 and CD1)


title: WT E16 basal cells, sample 1

description: Replicate 1 of 2.

source_name: Skin, WT

label_protocol: Performed by the Genomics Core Facility of MSKCC.


title: WT E16 basal cells, sample 2

description: Replicate 2 of 2.


title: sDf34

source_name: whole animal, mixed stage; Strain: BC1782


title: MTb-relE3 -24 hr - repeat 2 - mAdbID:77670

status: Public on Dec 15 2009

city: Rockville

first: Helena

middle: I

last: Boshoff

state: MD

address: 12441 Parklawn Drive

department: LIG

institute: NIAID/ NIH

description: mAdb experiment ID: 77670

hyb_protocol: Hybridization Protocol Other: For pre-hybrization, apply 40 ul of pre-hybridization buffer (5X SSC, 0.1% SDS, 1% BSA) to the array and incubate at 42°C for at least 30 minutes and up to an hour. Wash off the pre-hybridization solution by rapidly plunging the slide in distilled water for 2 minutes, then transfer slide to 100% isopropanol for 2 minutes. Allow slide to air dry completely prior to use. (Can spin dry if in a rush.) (NOTE: Do not exceed 1 hour after pre-hybridization/drying before setting up hybridization.) For hybridization, combine Cy3 and Cy5 labeled targets together (~11 ul recovered for each). Add 1 µl herring sperm DNA (8-10 ug/ul) and 0.2 ul tNRA (8-10 ug/ul). Denature target at 100°C for 1 minute, then snap cool on ice. (Final volume should be about 12 ul.) Make fresh 2X Formamide hybridization buffer (50% formamide, 10X SSC, 0.2% SDS) and warm to 42°C just before adding to samples. Add 12 ul of 2X F-hyb buffer to samples. Load 25 ul sample onto microarray. Add 20 ul of 3X SSC to wells in hyb chamber to maintain humidity. Incubate overnight (12-16 hours) at 42°C in water bath or hybridization oven. After hybridization of slides, wash slides for 2 minute in 2X SSC with 0.1% SDS (on rocking platform), for 2 minute in 1X SSC (with rocking), for 2 minutes in 0.2X SSC (on rocking platform), for 1 minute in 0.05X SSC, and spin for 3 minutes at 650 rpm to dry.

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.88;; 3.71 Temperature: 31.79

data_processing:
mAdb Data Processing Protocol (v. 2)
Calculation Method: After background correction, ratios were median centered based on signals greater than 100 in both channels and linear transformed to obtain the log and linear values given in the data table.

organism: Mycobacterium tuberculosis

source_name: MTb-relE3 - 24 hr

label_protocol:
Cy5 Labeling Protocol
Other: 4ug total RNA is converted to cDNA using Invitrogen random primers and superscript III at 48C in presence of 10-fold excess of Cy5-dCTP above unlabeled dCTP according to manufacturer's instructions.

extract_protocol:
Trizol MTb Extraction
Extraction method: Trizol-QIAgen RNeasy Kit
Other: Mycobacterium tuberculosis was rapidly harvested and cell pellet suspended in Trizol, subjected to 2-3 rounds of bead beating (0.1mm zirconia beads), phase separation performed with CHCl3 and RNA purified with the Quiagen RNeasy clean up kit using Qiagen on-column DNase to remove contaminating DNA.

treatment_protocol:
Treatment type: compound
Agent: Anhydrotetracycline
Treatment dose: 50 ng/ml (in ethanol at final 1uL/mL)
Treatment time: 24 hours
Treatment temperature: 37 C

value: H37Rv transformed with pTE-relE3

source_name: MTb-relE3 - 24 hr - control

label_protocol:
Cy3 Labeling Protocol
Other: 4ug total RNA is converted to cDNA using Invitrogen random primers and superscript III at 48C in presence of 10-fold excess of Cy3-dCTP above unlabeled dCTP according to manufacturer's instructions.

treatment_protocol:
Treatment type: compound
Agent: Ethanol
Treatment dose: 1 ul/ml
Treatment time: 24 hours
Treatment temperature: 37 C


title: MTb-relE1 - 6 hr - repeat 1 - mAdbID:77666

description: mAdb experiment ID: 77666

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.88;; 3.7 Temperature: 30.97

source_name: MTb-relE1 - 6 hr

treatment_protocol:
Treatment type: compound
Agent: Anhydrotetracycline
Treatment dose: 50 ng/ml (in ethanol at final 1uL/mL)
Treatment time: 6 hours
Treatment temperature: 37 C

value: H37Rv transformed with pTE-relE1

source_name: MTb-relE1 - 6 hr - control

treatment_protocol:
Treatment type: compound
Agent: Ethanol
Treatment dose: 1 ul/ml
Treatment time: 6 hours
Treatment temperature: 37 C


title: sDf28

source_name: whole animal, mixed stage; Strain: BC1289


title: Ezh2cKO E16 basal cells, sample 2

description: Replicate 2 of 2.

extract_protocol: Stratagene absolutely RNA kit


title: MTb-relE3 -24 hr - repeat 1 - mAdbID:95190

description: mAdb experiment ID: 95190

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.51;; 3.78 Temperature: 31.3


title: MTb-relE2 - 6 hr - repeat 3 - mAdbID:95193

description: mAdb experiment ID: 95193

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.52;; 3.77 Temperature: 30.81

source_name: MTb-relE2 - 6 hr

value: H37Rv transformed with pTE-relE2

source_name: MTb-relE2 - 6 hr - control


title: sDf29

source_name: whole animal, mixed stage; Strain: BC1381


title: MTb-relE1 - 6 hr - repeat 3 - mAdbID:95195

description: mAdb experiment ID: 95195

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.51;; 3.76 Temperature: 30.51


title: MTb-relE3 -6 hr - repeat 2 - mAdbID:77668

description: mAdb experiment ID: 77668

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.87;; 3.71 Temperature: 31.39

source_name: MTb-relE3 - 6 hr

source_name: MTb-relE3 - 6 hr - control


title: MTb-relE2 - 6 hr - repeat 2 - mAdbID:77667

description: mAdb experiment ID: 77667

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.87;; 3.71 Temperature: 31.24


title: MTb-relE3 -72 hr - repeat 4 - mAdbID:94896

description: mAdb experiment ID: 94896

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.16;; 3.48 Temperature: 33.71

source_name: MTb-relE3 - 72 hr

treatment_protocol:
Treatment type: compound
Agent: Anhydrotetracycline
Treatment dose: 50 ng/ml (in ethanol at final 1uL/mL)
Treatment time: 72 hours
Treatment temperature: 37 C

source_name: MTb-relE3 - 72 hr - control

treatment_protocol:
Treatment type: compound
Agent: Ethanol
Treatment dose: 1 ul/ml
Treatment time: 72 hours
Treatment temperature: 37 C


title: MTb-relE1 - 24 hr - repeat 2 - mAdbID:95194

description: mAdb experiment ID: 95194

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.52;; 3.77 Temperature: 30.66

source_name: MTb-relE1 - 24 hr

source_name: MTb-relE1 - 24 hr - control


title: MTb-relE1 - 6 hr - repeat 4 - mAdbID:81124

description: mAdb experiment ID: 81124

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.78;; 3.69 Temperature: 32.28


title: MTb-relE2 -72 hr - repeat 5 - mAdbID:94895

description: mAdb experiment ID: 94895

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.56;; 3.79 Temperature: 33.44

source_name: MTb-relE2 - 72 hr

source_name: MTb-relE2 - 72 hr - control


title: MTb-relE2 - 24 hr - repeat 3 - mAdbID:95192

description: mAdb experiment ID: 95192

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.51;; 3.77 Temperature: 30.97

source_name: MTb-relE2 - 24 hr

source_name: MTb-relE2 - 24 hr - control


title: MTb-relE1 - 72 hr - repeat 3 - mAdbID:94894

description: mAdb experiment ID: 94894

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.17;; 3.48 Temperature: 33.38

source_name: MTb-relE1 - 72 hr

source_name: MTb-relE1 - 72 hr - control


title: sDf53

source_name: whole animal, mixed stage; Strain: BC3461

source_name: whole animal, mixed stage; Strain: BC2842


title: MTb-relE3 -72 hr - repeat 2 - mAdbID:79645

description: mAdb experiment ID: 79645

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.79;; 3.77 Temperature: 30.32


title: MTb-relE3 -6 hr - repeat 3 - mAdbID:95191

description: mAdb experiment ID: 95191

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.5;; 3.77 Temperature: 31.15


title: MTb-relE3 -72 hr - repeat 5 - mAdbID:94800

description: mAdb experiment ID: 94800

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.16;; 3.48 Temperature: 33.71


title: MTb-relE2 - 24 hr - repeat 2 - mAdbID:77669

description: mAdb experiment ID: 77669

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.87;; 3.71 Temperature: 31.51


title: MTb-relE2 -72 hr - repeat 1 - mAdbID:77675

description: mAdb experiment ID: 77675

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.87;; 3.7 Temperature: 32.46


title: MTb-relE3 -72 hr - repeat 1 - mAdbID:77840

description: mAdb experiment ID: 77840

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.87;; 3.69 Temperature: 32.58


title: MTb-relE2 -72 hr - repeat 2 - mAdbID:81128

description: mAdb experiment ID: 81128

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.77;; 3.69 Temperature: 30.78


title: MTb-relE3 -72 hr - repeat 3 - mAdbID:81129

description: mAdb experiment ID: 81129

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.77;; 3.7 Temperature: 30.87


title: MTb-relE1 - 72 hr - repeat 1 - mAdbID:81127

description: mAdb experiment ID: 81127

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.76;; 3.69 Temperature: 30.72


title: MTb-relE2 -72 hr - repeat 4 - mAdbID:94799

description: mAdb experiment ID: 94799

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.56;; 3.79 Temperature: 33.44


title: MTb-relE3 -6 hr - repeat 4 - mAdbID:81126

description: mAdb experiment ID: 81126

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.78;; 3.69 Temperature: 32.4


title: MTb-relE1 - 6 hr - repeat 2 - mAdbID:79639

description: mAdb experiment ID: 79639

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.78;; 3.79 Temperature: 29.29

source_name: MTb-relE1 - 6 hr

source_name: MTb-relE1 - 6 hr - control


title: MTb-relE1 - 72 hr - repeat 4 - mAdbID:94798

description: mAdb experiment ID: 94798

scan_protocol: Creator: GenePix Pro 6.1.0.4 Scanner: GenePix 4000B [82425] ScanPower: 100;; 100 LaserPower: 3.17;; 3.48 Temperature: 33.38


title: MTb-relE2 - 24 hr - repeat 1 - mAdbID:81125

description: mAdb experiment ID: 81125

scan_protocol: Creator: GenePix Pro 4.0.1.17 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.78;; 3.69 Temperature: 32.61


title: MTb-relE2 -72 hr - repeat 3 - mAdbID:79644

description: mAdb experiment ID: 79644

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.78;; 3.77 Temperature: 29.96


title: MTb-relE3 -6 hr - repeat 1 - mAdbID:79641

description: mAdb experiment ID: 79641

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.76;; 3.77 Temperature: 30.39


title: TFII-TF6

status: Public on Dec 11 2009

city: Barcelona

first: Marc

last: Güell

address: C/ Dr.Aiguader 88

department: Systems Biology

country: Spain

institute: Centre for Genomic Regulation

hyb_protocol: Hybridization and Scanning (Axon GenePix 4000) has been carried out in the Genomic Core Facility of EMBL-Heidelberg.

data_processing: After background subtraction quantile (Boltad 2003) normalization was done using the bioconductor package marray (Yang 2005)

organism: Mycoplasmoides pneumoniae M129

source_name: pMT85-tuf->GntR-flag  line

label_protocol: 9μg of total RNA were used so as to carry out the reverse transcription using SuperScriptTM Indirect cDNA Labeling System from Invitrogen. This kit was used according manufacturer indication but two modifications were introduced in the protocol. Reverse transcription was carried out at 37ºC instead of 46ºC and the set of random hexamers (2uL of 2.5ug/uL) was used instead of polyT 20mers.

growth_protocol: Bacterial Strains and culture conditions: Mycoplasma pneumoniae is grown in 150 cm2 tissue culture flasks with 50mL of modified Hayflick medium with the following composition. The basic medium consisted of 18.4g of PPLO broth, 29.8g of HEPES,10 g glucose, 5mL of 0.5% phenol red, and 35mL of 2N NaOH per liter. Horse serum and penicillin were included to a final concentration of 20% and 100 U/mL, respectively.

extract_protocol: After growth , surface-attached cells are washed once with phosphate-buffered saline (PBS; 0.15M NaCl, 10mM sodium phosphate, pH 7.4) and immediately lysed in the cultivation flask by adding RLT buffer from the QiagenTM RNeasy Plus Mini Kit (Cat. Num. 74134). This isolation method is used for RNA extraction as it removes most RNAs smaller than 200 bases, thus preventing the synthesis of cDNA from tRNA. For cell lysis, 2mL of RLT buffer in presence of 20μL β-mercaptoethanol was used per cultivation flask. The purification is done according the manufacturers protocol.

tag: Strain M129

tag: whole organism


title: GC_1-48h

scan_protocol: Hybridization and Scanning (Axon GenePix 4000) has been carried out in the Genomic Core Facility of EMBL-Heidelberg.

source_name: M129 cells were collected after 2 days in culture


title: TFII-TF13

source_name: pMT85-tuf->relA-flag  line


title: SR-15'NV

source_name: Cells grown for 2 days, 0.5mg/ml DL-norvaline added for the indicated time points (15 min) to generate unloaded tRNAs


title: SR-SR

source_name: Amino acid starvation: stringent response in minimal medium, cells grown 2 days in rich medium, and changed for 15 min to minimal medium with (CtlSR) and without peptides and amino acids (SR)


title: GC_1-72h

source_name: M129 cells were collected after 3 days in culture


title: SR-60'NV

source_name: Cells grown for 2 days, 0.5mg/ml DL-norvaline added for the indicated time points (60 min) to generate unloaded tRNAs


title: SR-2dHS

source_name: 20 min heat-shock at 42C after 2 days in cluture (control is SR-0'Shx)


title: TFII-TF5

source_name: pMT85-tuf->hrcA-flag  line


title: SR-ctlSR


title: SR-5'NV

source_name: Cells grown for 2 days, 0.5mg/ml DL-norvaline added for the indicated time points (5 min) to generate unloaded tRNAs


title: SR-30'NV

source_name: Cells grown for 2 days, 0.5mg/ml DL-norvaline added for the indicated time points (30 min) to generate unloaded tRNAs


title: GC_1-24h

source_name: M129 cells were collected after 1 day in culture


title: MTb-relE1 - 24 hr - repeat 1 - mAdbID:77672

description: mAdb experiment ID: 77672

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.87;; 3.72 Temperature: 32.09

source_name: MTb-relE1 - 24 hr

source_name: MTb-relE1 - 24 hr - control


title: MTb-relE1 - 72 hr - repeat 2 - mAdbID:77674

description: mAdb experiment ID: 77674

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.87;; 3.7 Temperature: 32.34

source_name: MTb-relE1 - 72 hr

source_name: MTb-relE1 - 72 hr - control


title: SR-30'Shx

source_name: Repeat of Shx (cellls grown for 2 days, then 25mM serine hydroxamate added for 30 min to generated unloaded tRNAs)


title: MTb-relE2 - 6 hr - repeat 1 - mAdbID:79640

description: mAdb experiment ID: 79640

scan_protocol: Creator: GenePix Pro 4.0.1.22 Scanner: GenePix 4000B [83871] ScanPower: 100;; 100 LaserPower: 2.78;; 3.79 Temperature: 29.41

source_name: MTb-relE2 - 6 hr

source_name: MTb-relE2 - 6 hr - control


title: SR-15'Shx

source_name: Repeat of Shx (cellls grown for 2 days, then 25mM serine hydroxamate added for 15 min to generated unloaded tRNAs)


title: New_slides-d48h

source_name: 24h time point of the growth curve, but with thawed cells, not split


title: TFI-TF14

source_name: pMT85-tuf->SigA-flag  line


title: TFII-TF15

source_name: pMT85-tuf->dnaA-flag  line


title: TFI-TF17

source_name: pMT85-tuf->555-flag  line


title: TFI-TF4

source_name: pMT85-tuf->rpoE-flag  line


title: SR-0'Shx

source_name: Repeat of Shx (cellls grown for 2 days, then 25mM serine hydroxamate added for 0 min to generated unloaded tRNAs)


title: New_slides-d24h


title: TFI-aMM12

source_name: Cells adapted to minimal medium


title: TFI-6h

source_name: Cells (4 days in culture) split in fresh medium, collected after 6 hours (can go with GC-4)


title: TFI-2hC

source_name: Cells (4 days in culture) split in their own medium, collected after 2 hours


title: TFI-2hF

source_name: Cells (4 days in culture) split in fresh medium, collected after 2 hours


title: TFI-TF18

source_name: pMT85-tuf->526-flag  line


title: TFI-TF16

source_name: pMT85-tuf->626-flag  line


title: GC_11-B170d2

source_name: Growth curve of the non-adherent strain B170, grown with aeration for 2 days


title: TFI-TF12

source_name: pMT85-tuf->recA-flag  line


title: GC_11-B170d3

source_name: Growth curve of the non-adherent strain B170, grown with aeration for 3 days


title: TFI-TF8

source_name: pMT85-tuf->295-flag  line


title: TFI-TF10

source_name: pMT85-tuf->fur-flag  line


title: GC_11-B170d4

source_name: Growth curve of the non-adherent strain B170, grown with aeration for 4 days


title: TFI-TF1

source_name: pMT85 line


title: GC_10-Ac1.1

source_name: Cells were seeded directly in Hayflick plus 10mM Na+Acetate and samples were taking every day (1)


title: GC_7-GG3

source_name: Cells were changed to 1%glucose/1%glycerol after one day in culture and samples were taken after 48 hours


title: GC_10-NaCl1.1

source_name: Cells were seeded directly in Hayflick plus 60mM NaCl and samples were taking every day, 1 (control for other experiments)


title: GC_10-NaCl2.1

source_name: Cells were seeded directly in Hayflick plus 60mM NaCl and samples were taking every day,2 (control for other experiments)


title: GC_4-144h

source_name: M129 cells were collected after the indicated hours(144) in culture (this goes with GC_4)


title: GC_11-B170d1

source_name: Growth curve of the non-adherent strain B170, grown with aeration for 1 day


title: SHX-3xcells

source_name: Cells seeded at 3 times more density and collected after 24 hours


title: SHX-30Shx

source_name: Cells grown for 2 days, then 25 mM serine hydroxamate added for 30 min to generated unloaded tRNAs


title: SHX-5xcells

source_name: Cells seeded at 5 times more density and collected after 24 hours


title: GC_10-LpH1.1

source_name: Cells were seeded directly in Hayflick at pH 6.5 and samples were taking every day (1)


title: GC_10-LpH2.1

source_name: Cells were seeded directly in Hayflick at pH 6.5 and samples were taking every day (2)


title: GC_10-LpH3.1

source_name: Cells were seeded directly in Hayflick at pH 6.5 and samples were taking every day (3)


title: GC_4-120h

source_name: M129 cells were collected after the indicated hours (120) in culture (this goes with GC_4)


title: GC_10-Lac2.1

source_name: Cells were seeded directly in Hayflick plus 60mM Na+Lactate and samples were taking every day (2)


title: GC_10-Lac3.1

source_name: Cells were seeded directly in Hayflick plus 60mM Na+Lactate and samples were taking every day (3)


title: GC_10-Lac1.1

source_name: Cells were seeded directly in Hayflick plus 60mM Na+Lactate and samples were taking every day (1)


title: GC_10-Ac3.1

source_name: Cells were seeded directly in Hayflick plus 10mM Na+Acetate and samples were taking every day (3)


title: GC_10-NaCl3.1

source_name: Cells were seeded directly in Hayflick plus 60mM NaCl and samples were taking every day, 3 (control for other experiments)


title: SHX-aMan

source_name: Cells adapted to increasing concentrations of mannose


title: SHX-0Shx

source_name: Cells grown for 2 days, then 25 mM serine hydroxamate added for 0 min to generate unloaded tRNAs (control)


title: Starv-starv10h

source_name: M129 cells were grown for 3 days in rich medium (time 0h), then washed and changed to medium without glucose, and starved cells were collected at 10h


title: GC_10-Ac2.1

source_name: Cells were seeded directly in Hayflick plus 10mM Na+Acetate and samples were taking every day (2)


title: GC_7-GG4

source_name: Cells were changed to 1%glucose/1%glycerol after one day in culture and samples were taken after 72 hours


title: Starv-starv4h

source_name: M129 cells were grown for 3 days in rich medium (time 0h), then washed and changed to medium without glucose, and starved cells were collected at 4h


title: Starv-starv6h

source_name: M129 cells were grown for 3 days in rich medium (time 0h), then washed and changed to medium without glucose, and starved cells were collected at 6h


title: Starv-starv8h

source_name: M129 cells were grown for 3 days in rich medium (time 0h), then washed and changed to medium without glucose, and starved cells were collected at 8h


title: Starv-starv2h

source_name: M129 cells were grown for 3 days in rich medium (time 0h), then washed and changed to medium without glucose, and starved cells were collected at 2h


title: GC_7-GG2

source_name: Cells were changed to 1%glucose/1%glycerol after one day in culture and samples were taken after 24 hours


title: SHX-15Shx

source_name: Cells grown for 2 days, then 25 mM serine hydroxamate added for 15 min to generate unloaded tRNAs


title: SHX-aFru

source_name: Cells adapted to increasing concentrations of fructose


title: GC_6-Gly4

source_name: Growth curve changing the medium to rich medium -glucose + 1% glycerol after 24 hours in culture, and collecting cells after 3 days in glycerol as only carbon source. Day 1 is the control before glycerol.


title: GC_6-Gly2

source_name: Growth curve changing the medium to rich medium -glucose + 1% glycerol after 24 hours in culture, and collecting cells after 1 day in glycerol as only carbon source. Day 1 is the control before glycerol.


title: GC_6-Gly1

source_name: Growth curve changing the medium to rich medium -glucose + 1% glycerol after 24 hours in culture, and collecting cells after 1, 2, 3 days in glycerol as only carbon source. Day 1 is the control before glycerol.


title: GC_6-Gly3

source_name: Growth curve changing the medium to rich medium -glucose + 1% glycerol after 24 hours in culture, and collecting cells after 2 days in glycerol as only carbon source. Day 1 is the control before glycerol.


title: GC_4-60h

source_name: M129 cells were collected after 60h


title: GC_4-96h

source_name: M129 cells were collected after 96h


title: GC_4-84h

source_name: M129 cells were collected after 84h


title: GC_4-24h

source_name: M129 cells were collected after 24h


title: GC_4-12h

source_name: M129 cells were collected after 12h


title: TFI-TF7

source_name: pMT85-tuf->SpxA-flag  line


title: GC_5-spin1

source_name: M129 cells were grown in rich medium with constant shaking and samples were taken after 1 day in culture.


title: GC_5-spin2

source_name: M129 cells were grown in rich medium with constant shaking and samples were taken after 2 days in culture.


title: Synch_3-EtOH-0h

source_name: M129 cells grown for 3 days in dishes, 50 ug/ml cytochalasin B was added (or vehicle, Ethanol) in new medium, and after 24 hours, and a time 0 sample was taken


title: GC_4-72h

source_name: M129 cells were collected after 72h


title: Synch_3-CytoB-16hC

source_name: M129 cells grown for 3 days in dishes, 50 ug/ml cytochalasin B was added (or vehicle, Ethanol) in new medium, and after 24 hours, time points every 2 hours were taken  after chasing with new medium (16 hours)


title: GC_5-spin3

source_name: M129 cells were grown in rich medium with constant shaking and samples were taken after 3 days in culture.


title: GC_4-36h

source_name: M129 cells were collected after 36h


title: GC_5-spin4

source_name: M129 cells were grown in rich medium with constant shaking and samples were taken after 4 days in culture.


title: Synch_3-CytoB-12hC

source_name: M129 cells grown for 3 days in dishes, 50 ug/ml cytochalasin B was added (or vehicle, Ethanol) in new medium, and after 24 hours, time points every 2 hours were taken  after chasing with new medium (12 hours)


title: GC_4-48h

source_name: M129 cells were collected after 48h


title: Synch_3-EtOH-20hC

source_name: M129 cells grown for 3 days in dishes, 50 ug/ml cytochalasin B was added (or vehicle, Ethanol) in new medium, and after 24 hours, time points every 2 hours were taken  after chasing with new medium (20 hours)


title: Synch_3-CytoB-18hC

source_name: M129 cells grown for 3 days in dishes, 50 ug/ml cytochalasin B was added (or vehicle, Ethanol) in new medium, and after 24 hours, time points every 2 hours were taken  after chasing with new medium (18 hours)


title: Synch_3-CytoB-20hC


title: Synch_3-CytoB-10hC

source_name: M129 cells grown for 3 days in dishes, 50 ug/ml cytochalasin B was added (or vehicle, Ethanol) in new medium, and after 24 hours, time points every 2 hours were taken  after chasing with new medium (10 hours)