GSM8666028: 7013_Scc2G1Depleted_N2; Saccharomyces cerevisiae; OTHER
Micro-C was performed following published protocols[Costantino, 2020 #1788][Slobadyanyuk, 2022 #1888] with minor modifications. Yeast cultures were crosslinked with 3% formaldehyde for 15 minutes at 30 °C. The reactions were quenched by the addition of 250 mM glycine at 30 °C for 5 minutes with agitation. Cells were pelleted by centrifugation at 4000 rpm at 4 °C for 5 minutes and washed twice with water. Cells were then resuspended in Buffer Z (50 mM Tris-HCl pH 7.5, 1M sorbitol, 10 mM β-mercaptoethanol) and spheroplasted by addition of 250 mg/ml Zymolyase (100T) in a 30 °C incubator at 200 rpm for 40 to 60 minutes. Spheroplasts were washed once by 4 °C PBS and then pelleted at 4000 rpm at 4 °C for 10 minutes. Chromatin was further crosslinked by suspending pellets in PBS supplemented with 3 mM disuccinimidyl glutarate (ThermoFisher) and incubated at 30 °C for 40 minutes with gentle shaking before quenching by addition of 400 mM glycine for 5 minutes at 30 °C. Cells were pelleted by centrifugation at 4000 rpm at 4 °C for 10 minutes, washed once with ice-cold PBS and stored at −80 °C. Pellets were then treated as described[Costantino, 2020 #1788] until the de-crosslinking step. The De-crosslinking solution was added with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), vortexed intensively and centrifuged for 15 minutes at room temperature. The aqueous phase was purified on a ZymoClean column (Zymo Research) according to the manufacturer instructions. Dinucleosome-sized DNA fragments were purified and excised from a 3% NuSieve GTG agarose gel (Lonza) using the Zymoclean Gel DNA Recovery Kit (Zymo). 17 ul of recovered DNA were treated with an end polishing following End-It DNA End-Repair Kit (Lucigen) manufacturer instruction before being diluted in 125 μL of water and added with 150 μL of 2x BW buffer. Biotynilated DNAs were precipitated using 5 μL of Dynabeads™ MyOne™ Streptavidin C1 for 30 minutes at 25 °C on a nutator. Beads were washed twice in 1x TBW buffer at 55 °C and once in 10 mM Tris HCl pH 7.5. Beads were then treated following NEBNext® Ultra™ DNA Library Prep Kit for Illumina® procedure. Briefly, beads were resuspended in 30 μL of End prep master mix and incubated according to the manufacturer instruction, followed by a standard adaptator ligation and USER enzyme digestion protocol. Adaptors were then washed 2 times in 1x TBW at 55 °C followed by one wash in 10 mM Tris pH 7.5. Beads were resuspended in 20 μL of Zymo Elution Buffer and an on-bead PCR was performed (8 to 12 cycles). Two rounds of 0.85X AMPure cleanup were performed. DNA fragments size were assessed on a Tapestation. Library were sequenced using an Illumina NovaSeq 6000 sequencer (100 bp paired). (see Slobodyanyuk et al. 2022 https://doi.org/10.1007/978-1-0716-2497-5_4)