micrococcal nuclease (MNase) experiments was performed as previously described (Seymour et al., 2016 (link)). Neurospora was cultured in petri dishes in liquid medium for two days. Afterwards, the Neurospora mats were cut into discs and transferred into medium-containing flasks and were harvested at DD14. After crosslinking with 1% formaldehyde (Sigma-Aldrich, F8775-500ML) for 30 min at room temperature, nuclei were incubated with micrococcal nuclease (Takara, Cat#2910A) for 60 min at 37 °C before the reaction was stopped by addition of 10mM EDTA (Fluka, Cat#03609). Sequencing libraries were generated using 50 ng of DNA purified from the MNase-digested chromatin (Diagenode iDeal Library Preparation Kit, Cat#C05010020) and size-selected by agarose gel purification to ensure insert sizes close to that of DNA bound by mononucleosomes. After 75bp paired-end sequencing, the MNase-seq data were normalized by total number of reads.
F8775 500ml
Manufactured by Merck Group
F8775-500ML is a laboratory equipment product from Merck Group. It is a 500 milliliter container for laboratory use. The core function of this product is to provide a standard container for various laboratory applications, without further interpretation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Micrococcal nuclease, by Takara Bio (1 mentions)
Ideal library preparation kit, by Diagenode (1 mentions)
Inverted axiovert 200 m microscope, by Zeiss (1 mentions)
Triton x 100, by Avantor (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Apyrase, by New England Biolabs (1 mentions)
G8898 1kg, by Merck Group (1 mentions)
Bodipy 558 568, by Thermo Fisher Scientific (1 mentions)
Fm4 64fx, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Fv1000, by Olympus (1 mentions)
5 protocols using f8775 500ml
1
Mapping Neurospora Chromatin Structure
2
BiFC Vector Transfection in HEK293T Cells
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HEK293T cells were seeded on coverslips and transfected with different combinations of the described BiFC vectors using lipofectamine 2000 reagent (Invitrogen, 11668019). Each interaction was performed for all eight vector pair combinations, encoding each of two proteins tested for their interaction. At 36 h post transfection, cells were fixed with 3.76% formaldehyde in PBS at room temperature (Sigma, F8775-500ML) for 10 min then permeablized in 0.2% Triton X-100 (VWR, CA97062-208) for 2 minutes. Cells were then washed three times with 1X PBS, stained with Hoechst (1∶5000) in 1X PBS for 5 min, and then washed three times with 1X PBS. Cells were then examined using a Zeiss inverted Axiovert 200 M microscope and 40x/0.75 objective lens to detect Hoechst and YFP. At least ten random fields of cells were imaged for each combination. Channels were merged using ImageJ software (National Institutes of Health). Photoshop Elements 10 (Adobe) software was used to adjust brightness and contrast levels for individual images and assemble images into figures.
3
Chromatin Digestion and Sequencing
4
Cross-linking and TNF Stimulation for NicE-C and RNA-seq
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For cross-linking, cultured cells were collected by trypsin and then fixed at room temperature for 10 min with 1% formaldehyde (Sigma-Aldrich F8775-500ML). After cross-linking, 2.5 M Glycine (Sigma-Aldrich G8898-1KG) was added to the final 125 mM and incubated at room temperature for 10 min to quench the reaction. Fixed cells were pelleted and washed twice with ice-cold 1× PBS. After removing the supernatant, the fixed cells were stored at –80°C until used. TNF (Sino biological 10602-HNAE) was dissolved in water to a final concentration of 20 ng/µL. Cultured HeLa-S3 cells were washed once with 1× PBS, and fresh DMEM media supplemented with final 10 ng/mL TNF or water were added. Cells were then incubated for 60 min and subjected to NicE-C or RNA-seq.
5
Lipid Droplets and Membrane Staining in Drosophila Retina
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For LD staining, Drosophila heads were cut in half and the brain was removed to expose the retina underneath. Retinas were fixed in 3.7% formaldehyde (Sigma, F8775-500ML) in PBS for 15 min and washed 3 times in PBS1X. Retinas were incubated for 10min in BODIPY™ 558/568 (Invitrogen, D3835) to label lipid droplets, and FM™ 4-64FX (Invitrogen, F34653) to stain membranes. Retinas were then mounted in Vectashield (Vector Laboratories Ltd, H-1000) and imaged on Olympus FV1000.
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