Flash freezing and semi-automated precision grinding of fission yeast in liquid nitrogen. (a) Pellet of fission yeast cells (beige) in a centrifuge bottle. The cells were harvested by centrifugation, washed and pooled in water, then re-pelleted and drained. (b) Pellet resuspended using minimal volume of INCA buffer (see text), then transferred to tip-capped syringe. (c) Syringe inserted into tube adapter (gray) and centrifuged in horizontal swinging-bucket rotor. Arrow indicates horizontal rotation. (d) After centrifugation, the total (upper boundary of light-beige supernatant) and pellet (upper boundary of dark-beige cell pellet) volumes were recorded by direct reading from the syringe scale. (e) Supernatant removed and plunger re-installed. (f) Tip cap removed, and packed fission yeast swiftly extruded into liquid nitrogen (blue) as a continuous bead or "noodle". (g) Noodle broken into 3- to 7-mm pieces in liquid nitrogen. (h) Pieces loaded into pre-cooled (liquid nitrogen) motorized precision mortar grinder and ground in liquid nitrogen at the minimum pressure setting ("0"). Arrows indicate directions of rotation of the mortar and contents (white arrows) and pestle (black arrows). (i) When slurry of ground material appears homogenous, pressure setting is raised to pre-determined optimum ("0/1" in our case) until release of intact nuclei, as determined by fluorescence microscopy, is maximized. (j) Cryo-ground cells transferred to storage container for evaporation of liquid nitrogen at -80°C. Octagons denote steps at which samples may be safely stored at -80°C (initially only loosely capped to allow venting of the evaporating nitrogen). White-bordered octagons: -80°C storage optional; solid red octagon: storage required. (k) 200-μl pipette tip bevel-cut and bent for collecting samples of mortar contents for microscopy during grinding

Method-independent consistency of MNase cut sites and nucleosome positions within the fission yeast ade6 gene region. The horizontal axis and transcription diagram at the bottom of this figure are common to panels A and B. Thick blue bars represent the CoDing Sequences (CDS) of the indicated genes. The narrow green bars represent the 5' untranslated regions (5'-UTRs) of these genes, while the narrow red pointed bars represent their 3'-UTRs. (a) Histogram (vertical orange bars) displaying the frequencies (tags at the indicated position per million tags genome-wide) of 36-nt sequence tags in mononucleosomal DNA (LFN sample; Figure 2f and Additional File 1C), which map uniquely to the forward (upward scale) and reverse (downward scale) strands of a 4-kbp region of S. pombe chromosome 3 centered on the ade6 gene. (b) Profiles of relative MNase protection. For the orange (LFN sample) and green (LUN sample) lines, this is defined as the frequency, relative to the genome average (horizontal black, dotted line), with which the indicated nucleotide position is crossed by computer-generated 3'-extrapolations of forward- and reverse-strand sequence tags to 120 bp. The pink line shows relative MNase protection based on the microarray results published by Lantermann et al. [21]. The percent GC (150-bp window) over the region is indicated by the gray line and right-hand vertical axis. The vertical black arrows and lines mark the positions of MNase cuts mapped within the ade6 gene by Lantermann et al. [7] using the traditional indirect-end-labeling method

Micrococcal nuclease (MNase) digestion of chromatin within fission yeast nuclei and recovery of protected DNA. (a) DAPI fluorescence/phase contrast micrograph of log-phase S. pombe cells flash-frozen to -196°C then thawed in INCA buffer (see Materials and Methods). (b) Cryo-ground log-phase cells suspended and thawed in INCA buffer. The arrow marks an intact cell adjacent to a group of discrete liberated nuclei. (c) Oil-immersion micrograph of similarly-prepared stationary-phase nuclei. Arrows denote in-focus, liberated nuclei displaying the typical S. pombe tri-lobed DAPI fluorescence pattern. The ratio of liberated nuclei to cell debris seems lower here than in (B) because the microscope is focused on a thin (due to high magnification) plane containing settled cell walls, but many of the released nuclei are floating above that plane. (d) Gel-electrophoretic analysis of DNA recovered from MNase-digested nuclei of native ("Unfixed") and formaldehyde-treated ("HCHO-Fixed") stationary phase cells. "EF" indicates exogenous-Enzyme-Free control incubations. "+MNase" denotes incubation at 25°C with 150 units of MNase per ml for the indicated times. "Mkr bp" is a commercial 50-bp DNA-size-marker ladder. "Mkr Kbp" is a commercial 0.2- to 10-kbp DNA-size-marker ladder. (e) Gel-electrophoretic analysis of "mononucleosomal" DNA recovered from a band excised from a preparative-scale gel ("explant"). Twice as much DNA was loaded into the third and fourth lanes as into the second lane. "Dgst" indicates total DNA recovered from the parent MNase digest (12 min) of unfixed, stationary-phase nuclei; "t", "d" and "m" respectively mark the tri-, di-, and mononucleosomal DNA bands. "+Δ" signifies heat-denaturation of sample immediately prior to loading as an assay for internal nicking. (f) Gel-electrophoretic analysis of DNA recovered from a broad explant centered on the "mononucleosomal" band of a preparative-scale gel ("LFB": Log-phase, Fixed cells, Broad explant), then run on a second preparative gel from which the "mononucleosomal" band was re-sampled as a narrow explant ("LFN": Log, Fixed, Narrow).

Integrity of DNA replication intermediates (RIs) in fission yeast cryo-ground cells. (a) Schematic depiction (based on the pioneering study by Brewer and Fangman [25]) of Southern blot signals from two-dimensional (2D) gel-electrophoretic resolution of a restriction-endonuclease-digested, asynchronously-replicating DNA population. Axis labels indicate the primary resolution criteria in each electrophoretic direction, with font size conveying relative influence of given factors. Color-matched cartoon renderings depict the comparative structures and sizes of probe-targeted fragments responsible for specific features of the signal pattern. Green: linear fragments ("arc of linears"), 1N: migration position of single-length simple linear version of probe-targeted fragment; 2N: migration position of double-length, simple linear version of probe-targeted fragment; Yellow: fragments bearing single, externally-initiated replication forks ("Y arc"); Orange: fragments bearing two converging, externally initiated replication forks ("X line"); Red: fragments bearing diverging replication forks, initiated from a common internally located origin ("Bubble Arc"). (b) Autoradiogram from 2D analysis of the 3-kb fission yeast rDNA KpnI/HindIII digestion fragment using HMW DNA prepared from our 972 h-wild-type cryo-ground cells. (c) Left: Autoradiogram from 2D analysis of DNA from panel B after enrichment for RIs by Benzoylated Naphthoylated DEAE-cellulose chromatography. Center: Schematic representation of selective retention of bubble-bearing DNA fragments in Low-Gelling-Temperature (LGT) agarose (blue filaments) by topological capture during gel polymerization. Right: Autoradiograph from 2D analysis of topologically purified bubble-bearing fragments.