NextSeq 500 paired end sequencing; Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation
Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation
All strains were plated on YPAD overnight, day cultures were incubated at 30 Celsius in YPAD plus ampicillin, 3.2x10^5 cells were incubated in YPAD plus ampicillin overnight at 30 Celsius, 300ml cells OD 0.2  in YPAD plus ampicillin were grown to OD 0.6 (generally ~3 hours).   OD660 0.66 cells were arrested with 50ng/ml final α-factor for 2.5 hours at 30 Celsius and then checked for adequate schmooing under brightfield. G1 control samples were fixed with 1% formaldehyde followed by glycine quench as per below. Cells were filter washed with 800 ml YPAD+ampicillin (30 Celsius) in vacuum trap and Whatman 1.2um 100mm diameter membrane filters cat.10400821, and brought up in 350ml YPAD+ampicillin+50uM EdU (final).  50ml aliquots were harvested at defined time course and formaldehyde-fixed 10' room temperature shaking followed by addition of 2.25ml 2.5M Glycine for 5' shaking, and then placed on ice.  Cells were washed 3X with cold TBS (20mM Tris pH8.0, 150mM NaCl) and frozen at -80 Celsius. Frozen cell pellets were brought up in ice cold Spheroplast Digestion Buffer (1M Sorbitol, 50mM NaCl, 10mM Tris-HCl pH 7.5, 5mM MgCl2, 1mM CaCl2, and 0.075% v/v Nonidet p40 alternative supplemented with 1mM 2-mercaptoethanol, and 0.5mM of each of the following: Spermidine, Pepstatin, Aprotinin, benzamidine, E64, and ABSF) and lysed in a Mini-Bead Beater 8 ® at 4 degrees Celsius.  MNase was titrated to give 80% mononucleosomal DNA bands, typically 30 units for 15m at 37 degrees Celsius.  Samples were incubated overnight at 65 degrees Celsius with proteinase K and the supernatant was treated with RNase A for 1h at 37 degrees Celsius, followed by phenol-chloroform extraction and isopropanol precipitation.  Excized mononucleosome bands from agarose size-selection gel were purified using Freeze-n-Squeeze columns followed by phenol-chloroform extraction and cold ethanol precipitation.  A Click-iT ® NascentRNACapture Kit (Invitrogen, C10365) was used to biotinylate EdU-labelled nascent DNA, which was pulled down with Streptavidin MyOneTM Dynabeads ® (Invitrogen, 65601) as per Invitrogen protocols. DNA was blunt-ended, an A-overhang was added, and Illumina adapters were ligated on, with Agencourt Ampure XP bead washes after each step.  PCR amplification was performed with 18 or fewer cycles using Illumina barcoded primers and primer PE 1.0, followed by a final Ampure bead purification.
Experimental Factor: genotype
wild type genotype - Mat-a ura3::GPD-TK(Thymidine Kinase)::URA3 (5 copies) aur1::Padh1-hENT1::AUR1C ade2-1 trp1-1 leu2-3,112 his3-11,15 bar1::TRP1
Experimental Factor: stimulus
-factor block
Experimental Factor: time
33
Experimental Factor: protocol
MNase
Experimental Factor: immunoprecipitate
none