ip: Total Rap1 IP antibody: anti-Rap1 Y-300 (Santa Cruz, Catalog # sc-20167, Lot # A3003)
Growth protocol
Yeast were grown overnight in YPD (Yeast Extract 1%, Peptone 2%, Dextrose 2%) and used to inoculate 800 ml of YPR (Yeast Extract 1%, Peptone 2%, Raffinose 2%) to an OD600 of 0.2 (Genesys 20 Spectrophotometer) in a 4 L Erlenmeyer flask. These cells were grown for approximately 4 hours to an OD600 of 0.4 and subsequently arrested using 5 µM alpha-factor (400 µL of 10 mM, GenScript) until 95% of the yeast cells were unbudded (~3hrs). Cells were then induced by adding 40% galactose directly to the growing yeast to a final galactose concentration of 2%. At this time additional alpha factor was added (400 µL of 10 mM, GenScript). At time points 0, 10, 20, 30, 40, 50, 60, 90, 120, 150 minutes following galactose induction, 35ml of culture was taken at each time point and added immediately to 37% formaldehyde to a final concentration of 1% for 20 minutes of crosslinking. 13 ml were taken and washed once in sterile ice cold water, pelleted, and frozen in liquid nitrogen for subsequent RNA preparation.
Extracted molecule
genomic DNA
Extraction protocol
IP DNA isoloated from 1mg protein from WCE by immunoprecipitation (Lieb et al 2001, Nature Genetics)
Label
Cy3
Label protocol
IP and/or Input DNA was amplified using the GenomePlex Complete Whole Genome Amplification (WGA) kit (WGA2-50RXN, Sigma) and then reamplified using GenomePlex WGA Reamplification Kit (WGA3-50RXN, Sigma) using provided protocols. DNA was purified using Zymo columns according to the manufacturer's instructions (Zymo Research).
Yeast were grown overnight in YPD (Yeast Extract 1%, Peptone 2%, Dextrose 2%) and used to inoculate 800 ml of YPR (Yeast Extract 1%, Peptone 2%, Raffinose 2%) to an OD600 of 0.2 (Genesys 20 Spectrophotometer) in a 4 L Erlenmeyer flask. These cells were grown for approximately 4 hours to an OD600 of 0.4 and subsequently arrested using 5 µM alpha-factor (400 µL of 10 mM, GenScript) until 95% of the yeast cells were unbudded (~3hrs). Cells were then induced by adding 40% galactose directly to the growing yeast to a final galactose concentration of 2%. At this time additional alpha factor was added (400 µL of 10 mM, GenScript). At time points 0, 10, 20, 30, 40, 50, 60, 90, 120, 150 minutes following galactose induction, 35ml of culture was taken at each time point and added immediately to 37% formaldehyde to a final concentration of 1% for 20 minutes of crosslinking. 13 ml were taken and washed once in sterile ice cold water, pelleted, and frozen in liquid nitrogen for subsequent RNA preparation.
Extracted molecule
genomic DNA
Extraction protocol
1/20 of WCE for IP used as Input
Label
Cy5
Label protocol
IP and/or Input DNA was amplified using the GenomePlex Complete Whole Genome Amplification (WGA) kit (WGA2-50RXN, Sigma) and then reamplified using GenomePlex WGA Reamplification Kit (WGA3-50RXN, Sigma) using provided protocols. DNA was purified using Zymo columns according to the manufacturer's instructions (Zymo Research).
Hybridization protocol
Samples were hybridized by Roche-Nimblegen, Inc. Madison Wisconsin according to internal protocols.
Scan protocol
Samples were scanned by Roche-Nimblegen, Inc. Madison Wisconsin according to internal protocols.
Description
46641 A01
Data processing
Data was scaled subtracting the biweight mean from the log2 ratio for each probe.
