GSM4295147: Jurkat_Mnase-seq; Homo sapiens; MNase-Seq
Cell suspensions were transferred to 50 mL conical tubes and pelleted at 420g for 10 min. Cells were then resuspended in PBS at a density of 1x106 cells/mL for crosslinking with 0.5% methanol-free formaldehyde (ThermoFisher) at room temperature with rotation for 10 min. The reaction was then quenched with 150 mM glycine (PBS buffer pH 7.5) for 10 min at room temperature. Cells were then pelleted by centrifuging at 420g for 10 min at 4°C and then washed twice with 20 mL cold PBS each time. Nuclei were collected by lysing the cells in Farnham's lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl and 0.5% NP-40 freshly supplemented with 1mM PMSF and EDTA-free Protease inhibitor cocktail), washed once with cold MNase buffer (20 mM Tris-HCl pH 7.5, 15 mM NaCl, 60 mM KCl, 2 mM CaCl2) and then resuspended in MNase buffer at a concentration of 10x106 cells/mL before digestion. Micrococcal nuclease (New England BioLabs, M0247S) digestion was performed using 1100 U enzyme/106 cells at 37C for 10 min to achieve roughly 80% mono-nucleosome – 20% di-nucleosome populations. The reaction was interrupted with Stop buffer (20 mM EDTA pH 8.0, 20 mM EGTA pH 8.0, 0.4% SDS) and then centrifuged at 21,000g for 5 min at 4C. The supernatants were saved and the small white pellet discarded. Samples (100 μl) were mixed with 1 volume of 2X Proteinase K buffer (4 mM EDTA pH 8.0, 40 mM Tris-HCl pH 6.8, 1M NaCl and 1 mg/mL Proteinase K) for reverse crosslinking at 65°C for 16 hrs. After reverse crosslinking the samples were first extracted with 1 volume of phenol-chloroform-isoamylic alcohol (25:24:1 ratio) with centrifugation at 21,000g for 5 min at 4°C and later with 1 volume of chloroform-isoamylic alcohol (24:1 ratio). The aqueous phase was transferred to a 1.5 mL epitube and precipitated with 2.5 volumes of 100% cold Ethanol and 1/10 volume of 3M NaOAc with centrifugation at 21,000g for 15 min at 4°C. Samples were finally washed with 75% Ethanol, air dried for ~5 min and resuspended in 20 μl of water. DNA concentration was measured by Qubit/Nanodrop. About 3 μg of DNA were loaded onto a 1.5% DNA agarose gel to verify the expected nucleosomal size distribution (80% mono- and 20% di-nucleosome). The mono-nucleosome band (~150 bp size) was excised and gel cleaned up using DNA Clean & Concentrator kit (Zymo Research) following the manufacturer's instructions. DNA was eluted with 20 μl water (25 ng/μl final concentration). Replicate DNA samples were analyzed on high sensitivity DNA tape on Agilent 2200 TapeStation and used for library preparation. Library was prepared with ~375 ng of mono-nucleosomal DNA using the KAPA Hyper Prep Kit (KAPA Biosystems, KK8502) according to the manufacturer's instructions. For the PCR amplification step, we inputted ~7.5 ng and performed 9 cycles of amplification, obtaining 700 ng (~35 ng/μl). The quality control of the MNase-seq library was done on Agilent 2200 Tapestation. A single peak with average size of 306 bp (including ligated adapters) was observed. The MNase-seq DNA library was diluted to 4.2 nM for sequencing on an Illumina NextSeq 500 instrument.
GEO Accession
GSM4295147