GSM1905055: MATalpha deltaRE_whole 5C Rep3; Saccharomyces cerevisiae; OTHER
Crosslinked cells were lysed by grinding in a cold mortar and pestle [Hi-C] The yeast culture was crosslinked with 37% formaldehyde to a final concentration of 3% in the culture media for 20 min at 25˚C.  The crosslinking was quenched by adding 2X the volume of formaldehyde used of 2.5M Glycine to the culture and incubated culture for 5 min at 25˚C.  Cells were lyses by grinding in a cold mortar and pestle.  The chromatin was solubilized by adding 1% w/v SDS and Incubating at 65˚C water bath for 10 min.  Excess SDS was quenched using 10% v/v Triton X-100.  The chromatin was digested with HindIII at 37˚C overnight in a water bath.  Digested ends were filled in with biotin-14-dCTP with Klenow at 37˚C for 2 hr in a water bath.  The klenow was inactivated by Incubating in a 65˚C water bath for 20 min.  Crosslinked and digested fragments were ligated together in a dilute reaction inorder to favor intra-molecular ligation in a 16˚C water bath for 8 hr.  Crosslinkes were reversed by incubating the ligation mixture with proteinase K in a 65˚C water bath.  Ligation products were purified with phenol (pH 8.0):chloroform and precipitated with sodium acetate pH 5.2 and ethanol.  The precipitated DNA was resuspended in 1X TE and then the DNA was re-extracted with phenol (pH 8.0):chloroform and precipitated with sodium acetate pH 5.2 and ethanol.  The DNA pellet was dried thoroughly and excess salt was washed out of the solution using an amicon 30kDa spin column using 1X TE.  Then the sample was concentrated using a 30kDa amicon column.  RNA was degrade using DNase free RNase A at 37˚C for 1 hr.  The Hi-C library was quantified on a 0.8% agarose gel in 0.5X TBE by comparing it to known concentration standards.  The Hi-C efficiencey was accessed by first PCR amplifing a neighboring interaction in the Hi-C library.  This amplicon was split and the aliquots were digested with either HindIII, NheI, or HindIII and NheI at 37˚C overnight.  The digested products were quantified on a gel and the percent Hi-C efficiency was calculated as the percent digested with NheI divided by the percent digested in the NheI and HindIII combined reaction.  Biotin was removed from un-ligated ends using T4 DNA polymerase at 20˚C for 4hrs and then at 75˚C for 20 min to inactivate the enzyme. The DNA was sheared to 50-700bp using the Covaris S2 sonicator. The DNA ends were repaired using T4 DNA Polymerase, T4 Polynucleotide Kinase, and Klenow fragment of DNA Polymerase I at 20˚C for 30 min.  The DNA was purified with one Qiagen MinElute Column.  A dATP was added to the 3’ end of the molecules by using Klenow Fragment (exo-) at 37˚C for 30 min and then at 65˚C for 20 min to inactivate the enzyme.  The end repaired and A-tailed library was fractionated to 100-300bp using 0.9x and then 1.1x ampure XP extractions.  For each 1.0 ng of biotinylated ligation products, 1.0 µl of Dynabeads MyOne Strepavidin C1 beads were used to enrich the library.  Illumina PE adapters were ligated to the library using NEB Quick Ligase at room temperature for 15 min.  The library was amplified using Illumina primers PE1.0 and PE2.0 for as few cycles as possible.  Cycle number was determined by a titration experiment that revealed which cycle would produce enough library for sequencing but didn’t produce higher molecular weight artifacts.  The library was purified once more with 1.8x Ampure XP to remove primers and primer dimers.  The final library was quantified on a bioanalyzer.  The libraries were sequenced on either the GAII or HiSeq platforms. [5C] The yeast culture was crosslinked with a final volume of 3% formaldehyde (37% w/v, Fisher, Catalog #: BP531-500) in the culture media at 25˚C for 20mins with shaking.  The remaining formaldehyde was quenched with an excess of 2.5M glycine.  The yeast cells were harvested by centrifugation and lysed by grinding with a mortar and pestle in the presence of liquid nitrogen.  The chromatin was digested with HindIII (NEB, R0104S) and the resulting crosslinked restriction fragments were ligated with T4 DNA polymerase (Invitrogen, 15224-090) in a dilute reaction so that intra-molecular ligation was favored.  The crosslinking was reversed by incubating overnight at 65˚C in the presence of proteinase K (Invitrogen, 25530-015).  The chimeric DNA molecules were purified with a series of phenol:chloroform pH 8.0 (1:1) extractions and precipitated with 100% cold ethanol and 3M sodium acetate pH 5.2.  The RNA was degraded with RNase A (Roche, Catalog #: 10109169001) and the yield was quantified using molecular weight standards run on an agarose gel.  3C-PCR was performed on all 3C libraries with 3C PCR primers which represent a variety of genomic distances.  Libraries which showed the characteristic, exponential decay of 3C-PCR product abundance relative to genomic distance passed quality control.  For one 5C reaction, 4 million genome copies (53.3ng) worth of 3C library were mixed with 1fmol of each of the 5C probes in a 40 µl reaction.  The reaction was heated to 95˚C for 9mins to melt the DNA, then slowly cooled to 55˚C to allow for the 5C probes to anneal to the 3C ligation products for a minimum of 4hrs and a maximum of 16hrs.  The annealed probes were then ligated together using Taq DNA Ligase (New England Biolabs, M0208S) at 55˚C for 1hr.  The 5C products were amplified with a high fidelity polymerase (Amplitaq Gold, Life technologies, 4398813) and 1.6µM of each of the universal primers (Universal_forward: 5’ -/5Phos/CCTCTCTATGGGCAGTCGGTGAT – 3’, Universal_reverse: 5’ – /5Phos/CTGCCCCGGGTTCCTCATTCTCT – 3’).  Five 5C reactions for a total of 20 million genome copies were used to produce each 5C library.  The Illumina Paired-end sequencing adapters and primers were used to add the Illumina Paired-end sequences to the 5C molecules in order to prepare them for sequencing on either the Illumina GAII or HiSeq platforms.
GEO Accession
GSM1905055