6 protocols
Title: Affymetrix CEL analysis. Description:
normalization data transformation protocol
First we performed quantile normalization between replicate measurements and averaged the signal for each probe over the replicate intensities for the Glc7-FRB strain. For analysis of the wild-type control strain only one replicate was analyzed. ChIP enrichments were obtained by dividing ChIP intensities by the corresponding input intensities. The normalized ChIP signal at each nucleotide was calculated as the median signal for all probes overlapping this position. Profiles were smoothed using running median smoothing with a window half size of 75 bp. Normalized ChIP-chip profiles are provided as .gff files in this submission ( See additional zipped files in http://www.ebi.ac.uk/arrayexpress/files/E-MTAB-1528 ).
nucleic acid hybridization protocol
5.5 ug of fragmented and labeled DNA were hybridized to a high-density custom-made Affymetrix tiling array (PN 520055) at 45°C for 16 h with constant rotational mixing at 60 rpm in a GeneChip Hybridization Oven 640 (Affymetrix, SantaClara, CA). Washing and staining of the tiling arrays were performed using the FS450_0001 script of the Affymetrix GeneChip Fluidics Station 450. The arrays were scanned using an Affymetrix GeneChip Scanner 3000 7G.
nucleic acid labeling protocol
DNA samples were amplified and re-amplified with GenomePlex Complete Whole Genome Amplification 2 (WGA2) Kit using the Farnham Lab WGA Protocol for ChIP-Chip (http://www.genomecenter.ucdavis.edu/farnham/protocol.html). DNA quantity and quality control was performed with a ND-1000 Spectrophotometer (NanoDrop Technologies) and was usually larger than 1 ug. In addition, DNA quality was monitored by agarose gel electrophoresis. The re-amplification was performed in the presence of 0.4 mM dUTP (Promega U1191) to allow later enzymatic fragmentation. The enzymatic fragmentation, labelling, hybridization and array scanning were done according to the manufacturer instructions (Affymetrix Chromatin Immunoprecipitation Assay Protocol P/N 702238). Enzymatic fragmentation and terminal labelling were performed by application of the GeneChip WT Double-Stranded DNA Terminal Labeling Kit (P/N 900812, Affymetrix). Briefly, re-amplified DNA was fragmented in the presence of 1.5 uL uracil-DNA-glycosylase (10 U/uL) and 2.25 uL APE1 (100 U/uL) at 30°C for 1 h 15 min. The average fragment size was in the range of 50-70 bases as determined by automated gel electrophoresis on an Experion system (Bio-Rad Laboratories, Inc.) that allowed the analysis of small amounts of DNA. The fragmented DNA was then labelled at the 3’-end by adding 2 uL and 1 uL of terminal nucleotidyl transferase (TdT, 30 U/uL) and GeneChip DNA Labeling Reagent (5 mM), respectively.
For immunoprecipitation, 700 uL of chromatin sample was incubated with 16.5 uL antibody-coated and prewashed magnetic Protein G beads (Dynabeads Protein G, life technologies) at 4°C over night. Beads were prewashed 4 times with BSA/PBS (5mg/mL bovine serum albumine in 137 mM NaCl, 2,7 mM KCl, 8,1 mM Na2HPO4*2H2O, 1,4 mM KH2PO4 pH 7,4 with protease inhibitors), coated with the respective antibody for 30 minutes at 4 °C (100 µL anti-Tyr1-P (3D12), 25 µL anti-Ser2-P or 5µL anti-Rpb3 (1Y26, cat. no. W0012, neoclone) or 13.6 µg rabbit IgG (SIGMA) for TAP tags per chromatin sample) and washed again 3 times. After immunoprecipitation, beads were washed 5 times with wash buffer (100 mM Tris-HCl pH 7.5, 500 mM LiCl, 1% NP40, 1% sodium deoxycholat) and once with TE buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA). Immunoprecipitated chromatin was eluted for 5 minutes at 95°C with ChIP elution buffer (0.1M NaHCO3, 1% SDS). Eluted chromatin was digested with Proteinase K (Sigma) at 37°C for 2 hours and the reversal of crosslinks was performed at 65°C over-night. DNA was purified with the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer instructions. DNA samples were amplified and re-amplified with GenomePlex Complete Whole Genome Amplification 2 (WGA2) Kit using the Farnham Lab WGA Protocol for ChIP-Chip (http://www.genomecenter.ucdavis.edu/farnham/protocol.html).
growth protocol
Yeast cultures were grown in 400 mL YPD to OD600 ~0.65, split and incubated with equal volumes of either Rapamycin (1 ug/mL f.c. in DMSO) or DMSO at 30°C for another 60 minutes before formaldehyde crosslinking. Precooled lysis buffer additionally contained a phosphatase inhibitor cocktail (PhosSTOP, Roche). Cell disruption by bead beating was performed for 2 hours.