GSM8141177: spn1 K192N 32U MNase; Saccharomyces cerevisiae; MNase-Seq
Yeast cultures (3 ml) were grown to OD600 = 0.7-1.0 at 30°C, then used to inoculate yeast cultures (400 ml) grown to OD600 = 1.0 at 28°C. Cells are centrifuged at 5000 x rpm for 10 minutes at 4°C and the supernatant is decanted. The cell pellets are resuspended in 30 ml of sorbitol buffer (50mM Tris-Cl pH 7.5, 1M sorbitol, 10mM MgCl2) containing 10mM 𝛽-mercaptoethanol (𝛽ME) and 1mM phenylmethylsulfonyl fluoride (PMSF). The cell pellets were resuspended in sorbitol buffer with 𝛽ME and PMSF and centrifuged at max rpm for 5 minutes at 4°C twice, and the cell pellets were weighed afterward. Pellets were then suspended in 0.66ml sorbitol buffer with 𝛽ME and PMSF per gram of cells. 20 µl of the cell suspension was set aside for monitoring cell wall digestion, and the remainder was incubated at 30°C with gentle agitation for 10 minutes.  Spheroplasting was performed by adding 0.34 ml of Zymolase (10mg/ml) per gram of cells to the sorbitol-cell suspension, followed by incubation at 30°C. Spheroplasting was monitored by determining the OD600 of 4 µl of cell suspension with or without Zymolase combined with 4 µl of 1% SDS and 992 µl of ddH2O. When the OD600 of the cells with Zymolase was ~10% of the cells without Zymolase, sorbitol buffer with PMSF was slowly added until the final volume is 30 ml. Spheroplasts were collected by centrifugation at max rpm for 5 minutes, followed by decanting of the supernatant. The spheroplasts were washed in 5 ml of sorbitol buffer two additional times, then suspended in Ficoll buffer (18% Ficoll 400, 20mM Tris-Cl pH 7.5, 0.5mM MgCl2). Each spheroplast suspension was transferred to a 50 ml Potter-Elvehjem homogenizer and homogenized on ice with six up-down even strokes with a handheld drill at max rpm. The suspensions were transferred to chilled 25 ml centrifuge tubes, and the remaining cells were pelleted at 6400 x rpm for 5 minutes at 4°C. The supernatant containing yeast nuclei was transferred to new centrifuge tubes and pelleted at 15,000 x rpm at 4°C. Nuclei were suspended in 10 ml of MNase digestion buffer (20 mM Tris-Cl pH 8.0, 1.5 mM CaCl2, 150 mM NaCl, and 1 mM PMSF). The OD600 of the nuclei suspension was determined by a 1:10 dilution in digestion buffer, which should be ~0.2 for nuclei from 1 gram of cells. Nuclei were pelleted at 15,000 x rpm for 30 minutes at 4°C and suspended in digestion buffer at a ratio of 2.4 ml for OD600 = 0.2. 400 µl aliquots of the nuclei suspension were prepared for MNase digestion and incubated at 37°C for 10 minutes. Nuclei were digested with a range of MNase concentrations (0U, 32U, 64U, 128U, and 256U) at 37°C for 10 minutes. 100 µl of stop solution (50mM EDTA, 2.5% SDS, 0.25mg/ml Proteinase K) was added to each reaction, then samples were incubated at 37°C overnight. Samples were extracted twice with phenol:chloroform, once with chloroform, and precipitated with ethanol. The DNA was washed with 70% ethanol, dried at room temperature, then suspended in 100 µL of TE buffer (pH 7.5) and 3 µL of RNaseA (10 mg/mL). Reactions were incubated overnight at 37°C, followed by ethanol precipitation, a wash with 70% ethanol, and drying at room temperature. The precipitated DNA was resuspended in 100µl of TE buffer (10mM Tris-Cl, pH 8.0 and 1mM EDTA, pH 8.0), and 10µl was subjected to agarose gel electrophoresis. MNase-protected DNA fragments were made into sequencing libraries using the single stranded library protocol as previously described (Ramani et al., 2019; Snyder et al., 2016). In brief, 10ng DNA was dephosphorylated using FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific cat. EF0651), denatured, and incubated overnight with CircLigaseII (Lucigen cat. CL9025K) and 0.093-0.125 µM biotinylated CL78 primer (Snyder et al., 2016) at 60ºC with shaking every 5 minutes. DNA fragments were then denatured and bound to magnetic streptavidin M-280 beads (Invitrogen cat. 11205D) for 30 minutes at room temperature with nutation. Beads were washed, and second-strand synthesis was performed using Bst 2.0 DNA polymerase (NEB cat. M0537) with an increasing temperature gradient of 15-31ºC with shaking at 1750 rpm. Beads were washed, and a 3' gap fill was performed using T4 DNA polymerase (Thermo Scientific cat. EL0011) for 30 minutes at room temperature. Beads were washed, and a double-stranded adapter was ligated using T4 DNA ligase (Thermo Scientific cat. EP0062) for 2 hours at room temperature with shaking at 1750 rpm. Beads were washed and resuspended in 30 µL 10 mM TET buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.05% Tween-20). DNA was denatured at 95ºC for 3 min, and DNA libraries were collected after immediate magnetic separation. Quantitative real-time PCR was performed on DNA libraries using iTAQ Supermix (Bio-Rad cat. 1725124), and Ct values were used to determine the number of PCR cycles needed to amplify each library. PCR was performed with KAPA HiFi DNA polymerase (Kapa Biosystems cat. KK2502) using barcoded indexing primers for Illumina. Primer dimers were removed from the libraries using AMPure beads (Beckman Coulter cat. A63881). Libraries were eluted in 0.1X TE, and concentrations were determined using Qubit. The length distribution of each library was assessed by the Agilent Bioanalyzer using the D1000 or HSD1000 cassette. Libraries were sequenced for 150 cycles in paired-end mode on the NovaSeq 6000 system at the University of Colorado Cancer Center Genomics Shared Resource.