-Platform: amino-silane coated glass slides (GAPS II, Corning) -S. cerevisiae intergenic regions amplified from S288C genomic DNA (ResGen) using the intergenic region primer oligonucleotides (ResGen) (Harismendy et al. EMBO J. 22(18): 4738-4747, 2003). The primers allow the amplification of the sequence located on either side of elements such as open reading frames, tRNAs, small nuclear RNAs, Ty elements, solo δ, etc. A complete description of the primers can be obtained on the web site (ftp://ftp.resgen.com/pub/genepairs/yeast_intergenic). -The PCR products were purified and spotted using an automated arrayer (MicroGrid II, BioRobotics)
Experiment design -Type of experiment: genome wide binding analysis -Experimental factor: genotype (Wild-Type, Fhl1-myc, Ifh1-myc); drug (rapamycin) -Number of hybridization performed in the experiment: 15 (four conditions with three to five biological replicates each) -Reference used for each condition: ChIP from the wild-type strain -Quality control: one dye-swap per condition
Samples used, extract preparation and labeling -The origin of the biological sample: o Species: S. cerevisiae o Cell type: (1) W303; (2) W303+IFH1-13myc-TRP1MX6;(3) W303+FHL1-13myc-HIS3MX6. -Growth conditions: log phase (2x107/mL) at 30ºC in YPD medium; when indicated rapamycin was added at the final concentration of 200 ng/mL for 90 min. -Cross-linking for 30 min at 30ºC -Chromatin immunoprecipitation: Dynabeads M280 coupled to sheep anti-mouse IgG or sheep anti-rabbit IgG and mouse monoclonal antibodies against the myc epitope (culture supernatant of 9E10) were used for immunoprecipitation according to standard techniques. -Labeling protocol(s): each IP was blunt-ended, ligated to linker DNA, then subjected to Ligation Mediated (LM)-PCR in the presence of amino-allyl dUTP. Labeling was performed for 1 hour at room temperature in the presence of NHS-ester Cy3 or Cy5 in 0.1 M Sodium Carbonate. The labeling reaction was stopped with 2 M Hydroxylamine and the labeled DNA purified.
Hybridization procedures and parameters -Standard protocols were used
Measurement data and specifications -Software for scanning: GenePix Pro 4.0 (Axon Instruments, Inc.) -Scanner: GenePix 4000A (Axon Instruments, Inc.) Keywords: dose response
meiosis-specific gene required for meiotic recombination
iYLR029C
2.00
4.54E-07
YLR030W
RPL15A
hypothetical ORF
iYJL192C
3.31
7.44E-07
RPS14B
SOP4
ER to Golgi transport
iYBR189W
4.30
7.71E-07
YBR190W,RPL21A
-
-
iYMR142C
5.02
7.95E-07
RPS16A
RPL13B
-
iYER116C
4.62
1.88E-06
RPL23B
SLX8
DNA recombination
iYKL065C
2.06
2.54E-06
MNR2
YET1
unknown (putative magnesium transporter) / yeast endoplasmic reticulum 24 kDa transmembrane protein (homolog of BAP31)
iYNL307C
2.24
2.55E-06
MRPS18
MCK1
Mitochondrial ribosomal protein of the small subunit / 43.1 kDa serine/threonine/tyrosine protein kinase (glycogen synthase kinase 3 activity); chromosome segregation; entry in meiosis; NHEJ
iYGL189C
4.55
4.14E-06
-
RPS26A
-
iYLR047C
5.58
4.16E-06
RPS0B
YLR047C
hypothetical ORF
iYJL136C
5.73
4.71E-06
YJL135W,LCB3
RPS21B
hypothetical ORF/ sphingolipids biosynthesis
iYDR416W
3.18
6.14E-06
RPL12B
-
-
iYOL120C
2.02
7.36E-06
-
RPL18A
-
iYPL249C-A
2.57
7.50E-06
-
RPL36B
-
iYJR122W
6.15
7.57E-06
RPS5
-
-
Total number of rows: 100
Table truncated, full table size 5 Kbytes.
FHL1_Rapa_enriched header descriptions
Intergenic
Ratio
P-value
ORFc
ORFw
GO (except RPGs)
Data table
Intergenic
Ratio
P-value
ORFc
ORFw
GO (except RPGs)
iYLR184W
1.99
2.20E-10
RPL37A
-
-
iYJL136C
3.03
8.33E-10
YJL135W,LCB3
RPS21B
hypothetical ORF/ sphingolipids biosynthesis
iYIL133C
4.62
6.60E-09
-
RPL16A
-
iYHL033C
2.10
1.09E-08
-
RPL8A
-
iYML073C
5.21
2.47E-08
-
RPL6A
-
iYGL136C
2.67
2.52E-08
RPL1B
MRM2
mitochondrial 21S rRNA methyltransferase
iYER116C
3.58
3.51E-08
RPL23B
SLX8
DNA recombination
iYLR047C
4.98
5.13E-08
RPS0B
YLR047C
hypothetical ORF
iYGR117C
4.43
5.43E-08
RPS23A
YGR117C
unknown
iYPL249C-A
2.16
6.13E-08
-
RPL36B
-
iYFR031C-A
4.59
7.34E-08
-
RPL2A
-
iYGL104C
3.34
7.63E-08
RPL28
VPS73
vacuolar targeting
iYIL069C
3.08
8.90E-08
-
RPS24B
-
iYDR449C
2.29
9.21E-08
RPS18A
UTP6
part of small ribosomal subunit processosome
iYOR292C
3.90
1.25E-07
RPS10A
YOR292C
hypothetical ORF
iYML026C
4.66
1.27E-07
-
RPS18B
-
iYML025C
3.63
1.43E-07
RPS17A
YML6
mitochondrial ribosomal protein of the large subunit
iYDL076C
4.28
1.43E-07
RPL31A
YDL076C
putative histone deactylase
iYEL054C
3.07
2.47E-07
-
RPL12A
-
iYHR141C
4.66
2.59E-07
CHS7
RPL42B
ER to Golgi transport
Total number of rows: 99
Table truncated, full table size 5 Kbytes.
IFH1_untreated_enriched header descriptions
Intergenic
Ratio
P-value
ORFc
ORFw
GO (except RPGs)
Data table
Intergenic
Ratio
P-value
ORFc
ORFw
GO (except RPGs)
iYIL133C
3.00
7.25E-09
-
RPL16A
-
iYHR141C
4.39
9.26E-08
CHS7
RPL42B
ER to Golgi transport
iYMR142C
2.75
2.10E-07
RPS16A
RPL13B
-
iYIL069C
3.72
2.47E-07
-
RPS24B
-
iYOR234C
3.05
3.15E-07
snr17a
RPL33B
snoRNA
iYIL052C
3.72
3.41E-07
-
RPL34B
-
iYPL144W
1.92
4.63E-07
RPL33A
-
-
iYJR122W
3.79
4.93E-07
RPS5
-
-
iYDR416W
2.41
8.80E-07
RPL12B
-
-
iYJL137C
2.09
1.03E-06
-
GLG2
self-glucosylating initiator of glycogen synthesis
iYDL184C
2.26
1.23E-06
-
RPL41A
-
iYBL093C
2.65
1.32E-06
RPL32
ROX3
RNA polymerase II holoenzyme component
iYOR292C
2.05
1.90E-06
RPS10A
YOR292C
hypothetical ORF
iYOR312C
3.30
1.91E-06
-
RPL20B
-
iYOL128C
1.81
2.19E-06
RPL25
YGK3
glycogen synthase kinase 3 activity
iYHR203C
2.32
3.32E-06
MNL1
RPS4B
ER alpha-mannosidase-like involved in ERAD
iYNL096C
1.90
3.40E-06
-
RPS7B
-
iYKL006C-A
2.34
4.70E-06
RPL14A
SFT1
v-SNARE (Golgi)
iYML025C
2.70
4.93E-06
RPS17A
YML6
mitochondrial ribosomal protein of the large subunit
iYNL302C
3.52
6.65E-06
-
RPS19B
-
Total number of rows: 92
Table truncated, full table size 4 Kbytes.
IFH1_Rapa_enriched header descriptions
Intergenic
Ratio
P-value
ORFc
ORFw
GO (except RPGs)
Data table
Intergenic
Ratio
P-value
ORFc
ORFw
GO (except RPGs)
iYHR136C
2.84
1.01E-06
ARO9
SPL2
aromatic amino acid aminotransferase II / identical to PHO81 (inhibitor of of a cyclin (Pho80p)-dependent protein kinase (Pho85p) complex (Cdk))