GSM8640790: MNase-seq of Mmyco strain, 20min; Saccharomyces cerevisiae; MNase-Seq
Cell pellets were thawed on ice and resuspended in 1mL 1xPBS supplemented with 27ul 37% formaldehyde. Cells were fixed for 20min at room temperature on rotation. 200ul of 2.5M Glycine was added for a further 5 min incubation. Cells were centrifuged at 2500g, 4C for 5 min. Pellets were gently washed with cold 1xPBS before resuspension in 500ul of lysis solution (Sorbitol 1M, EDTA 10mM, Zymo100T 250ug) and incubation at 37C with gently shaking for 30min/45min or until complete spheroplasting. Cells were gently washed with cold 1xPBS and resuspended in 50ul 1X Nuclease Digest buffer plus an increasing amount of Mnase (NEB, M0247S) enzyme starting with 0.2ul. Digestion was performed at 22C in a shaker set at 1250rpm for 15min. 5ul of 0.5M EGTA and 3ul of 20% SDS were added for incubation at 22C in a shaker set at 1250rpm for 5 min. 65ul of TES buffer (50 mMTris-HCl, pH 8.0; 10 mM EDTA; 1% SDS) was added and samples were de-cross-linked by incubation at 65°C overnight. RNA was degraded by incubation with 2µL RNase A (10 mg/mL) for 1h at 37°C. Proteins were removed by incubation with 10µL of proteinase K (18 mg/mL) for 2h at 65°C. DNA was purified using Zymo Research DNA Clean and Concentrator™-5 Kit. Mnase digestion was verified on a Bioanalyzer System using HS DNA kit. DNA was purified using 1.2x volume of AmpureXP beads and librairies prepared with Invitrogen TM Collibri TM PS DNA Library Prep Kit for Illumina following manufacturer instructions. Paired-end sequencing on an Illumina NextSeq500 (2x35 bp) was performed.