A single centromeric nucleosome mediates attachments to a single microtubule in the thermotolerant yeast K. marxianus
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion ETD, Orbitrap Eclipse
ORGANISM(S): Kluyveromyces Marxianus (ncbitaxon:4911)
SUBMITTER: 
Susan Biggins
PROVIDER: MSV000095817 | MassIVE |
REPOSITORIES: MassIVE
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2024-10-16 | MSV000096113 | MassIVE
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2019-10-28 | PXD013080 | Pride
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2025-02-14 | MSV000097121 | MassIVE
Project description:Immunoaffinity Purifications of Human Argonaute Twelve hours after transfections with mock or with miR-124, we washed each 15-cm plate once with phosphate-buffered saline (usually two plates were used per IP), then added 1 ml of 4 C lysis buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM Na-EDTA [pH 8.0], 0.5% Nonidet P-40, 0.5 mM DTT, 10 ul protease inhibitor cocktail [Pierce Cat# 78437], 100 U/ml SUPERaseIn [Ambion Cat# AM2694]). Following a 30-min incubation at 4 C, we scraped the plates, combined the lysates, and then spun them at 4 C for 30 min at 14,000 RPM in a microcentrifuge. We collected the supernatant and filtered it through a 0.45-um syringe filter. We froze an aliquot of lysate in liquid nitrogen for reference RNA isolation. We then added 0.22 mg/ml heparin to the lysate. We mixed the lysate with 2.5 mg of Dynal m-280 streptavidin beads (250 ul from original storage solution) coupled to biotinylated 4F9 ago antibody (~12.5 ug), which we equilibrated immediately prior to use by washing twice with 1 ml of lysis buffer. We incubated the beads with the lysate for 2 h at 4 C and then washed the beads twice with 1.25 ml of ice-cold lysis buffer for 5-min each. Five percent of the beads were frozen for SDS PAGE analysis after the second wash. RNA was extracted directly from the remaining beads using lysis buffer from Invitrogen's Micro-to-Midi kit (Invitrogen Cat# 12183-018). We purified RNA from the lysate and RNA extracted from the beads with the Micro-to-Midi kit as per vender's instructions, except that the percentage isopropanol used for binding to the column was 70%, instead of 33%, to promote the binding of small RNAs. RNA was amplified with ambion kit 1755. RNA from total cell lysate was labeled with cy3 and IPd RNA was labeled with cy5
2009-11-10 | GSE18834 | GEO
Project description:We determined genome-wide nucleosome occupancy in mouse embryonic stem cells and their neural progenitor and embryonic fibroblast counterparts to assess features associated with nucleosome positioning during lineage commitment. Cell type and protein specific binding preferences of transcription factors to sites with either low (e.g. Myc, Klf4, Zfx) or high (e.g. Nanog, Oct4 and Sox2) nucleosome occupancy as well as complex patterns for CTCF were identified. Nucleosome depleted regions around transcription start and termination sites were broad and more pronounced for active genes, with distinct patterns for promoters classified according to their CpG-content or histone methylation marks. Throughout the genome nucleosome occupancy was dependent on the presence of certain histone methylation or acetylation modifications. In addition, the average nucleosome-repeat length increased during differentiation by 5-7 base pairs, with local variations for specific genomic regions. Our results reveal regulatory mechanisms of cell differentiation acting through nucleosome repositioning. For chromatin immunoprecipitation, for each sample, 1 x 106 cells were cross-linked with 1% PFA and cell nuclei were prepared using a swelling buffer (25 mM Hepes pH 7.8, 1 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT). Chromatin was sheared to mononucleosomal fragments. After IgG preclearance the sheared chromatin was incubated with 4 µg of either a H3K9ac (Abcam, ab4441), a H3K27ac (Abcam, ab4729), or a H3K9me3 (Abcam ab8898) antibody over night. After washes with sonication (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% N-lauroylsarcosine, 0.1% Na-deoxycholate), high-salt- (50 mM Hepes pH 7.9, 500 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS), lithium- (20 mM Tris-HCl pH 8.0, 1mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate) and 10 mM Tris-HCl, chromatin was eluted from the protein G magnetic beads and the crosslink was reversed over night. After RNase A and proteinase K digestion, the DNA was purified and subsequently cloned into a multiplexed Illumina library according to standard protocols. Sequenced 50 bp reads were mapped with bowtie and subsequently clustered with MACS 55 implemented in the Genomatix software suite (Genomatix, Munich, Germany) using a p-value of 10-5.
2012-10-21 | E-GEOD-40951 | biostudies-arrayexpress
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2021-07-08 | PXD019017 | Pride
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2010-06-20 | E-GEOD-18834 | biostudies-arrayexpress
Project description:The high level of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes suggests that mechanisms interpreting 5hmC in the central nervous system (CNS) may differ from those present in embryonic stem cells. Here we present quantitative, genome-wide analysis of 5hmC, 5- methylcytosine (5mC) and gene expression in differentiated CNS cell types in vivo. We report that 5hmC is enriched in active genes, and that surprisingly strong depletion of 5mC is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG binding protein 2 (MeCP2) as the major 5hmC binding protein in the brain, and demonstrate that MeCP2 binds 5hmC and 5mC containing DNA with similar high affinities. The Rett Syndromecausing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell specific epigenetic mechanism for regulation of chromatin structure and gene expression. Isolation and sorting was performed as described in (Kriaucionis and Heintz, 2009). Briefly, cerebella were dissected as described above and homogenized in homogenization medium (0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8, 0.15 mM spermine, 0.5 mM spermidine, EDTA-free protease inhibitor cocktail (Roche) using loose (A) and tight (B) glassglass dounce.Homogenate was supplemented with 50% iodixanol (OptiPrep, Axis-Shield, Scotland), 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8; and laid on a 29% iodixanol cushion. GC nuclei from WT and Mecp2-null mice were sorted by selecting a specific FSC/SSC population identified from GC EGFP+ mice. We observed that this FSC/SSC gated population in GC EGFP+ animals resulted >92% of the sorted nuclei being EGFP positive. Thus, the gate selected allows us to enrich the sample from 65-70% to 92-96% on GC cells. 5hmC was pulled down as described (Song et al., 2010). After purification DNA was amplified as described in TruSeq DNA Sample kit. 1-0.5 M-NM-<g of DNA was used for each experiment. Sonicated DNA was end-repaired following by ligation to Illumina paired end sequencing adapters (Illumina, PEM-bM-^@M-^P 102M-bM-^@M-^P 1003).), following by amplification with Illumina primers. Input samples were produced for each cell types in both procedures 5hmC enriched were then sequenced using Illumina platform obtaining more than 50 x10-6, 36- bp single-end reads per sample. Reads were aligned to mm9 mouse genome assembly using Bowtie v0.12.7 (-m1 --best). Two biological replicas were done for each of the cell types Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 M-NM-<g/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 M-CM-^W g, 4 M-BM-0C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 M-CM-^W g to pellet insoluble material. At this point, 30 M-NM-<L of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4M-BM-0C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 M-NM-<g/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) with in-column DNase digestion. RNA quantity and quality were determined with a Nanodrop 1000 spectrophotometer (Wilmington, DE) and Agilent 2100 Bioanalyzer using RNA 6000 Pico Chip. 4 biological replicas was produced per each cell type. 10 ng of total RNA from cerebellum were converted to cDNA using the NuGEN Ovation RNA-Seq (NuGEN,San Carlos,CA,USA) following manufacturersM-bM-^@M-^Y instructions. The Single Primer Isothermal Amplification (SPIA) method used in this protocol allows the amplification of RNA target in double stranded cDNA under standardized conditions that markedly deplete rRNA without preselecting mRNA. cDNA obtained was quality scored by RNA 6000 PicoChip for Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 1 M-NM-<g of cDNA per sample was sonicated using Covaris-S2 system (Covaris Inc., Woburn, MA, USA), cDNA fragments of 200 bp were end-repaired and adapters were ligated for HiSeq 2000 (Ilumina Inc., San Diego, CA, USA) technology using TruSeq DNA Sample kit (Illumina) and following manufacturer`s instructions. Quality of libraries was assesed using HT DNA High Sensitivity Chip (Agilent) for 2100 Bioanalyzer. Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 M-NM-<g/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 M-CM-^W g, 4 M-BM-0C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 M-CM-^W g to pellet insoluble material. At this point, 30 M-NM-<L of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4M-BM-0C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 M-NM-<g/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) with in-column DNase digestion. RNA quantity and quality were determined with a Nanodrop 1000 spectrophotometer (Wilmington, DE) and Agilent 2100 Bioanalyzer using RNA 6000 Pico Chip. 4 biological replicas was produced per each cell type. 10 ng of total RNA per IP or input sample were converted to cDNA using the NuGEN Ovation RNA-Seq (NuGEN,San Carlos,CA,USA) following manufacturersM-bM-^@M-^Y instructions. The Single Primer Isothermal Amplification (SPIA) method used in this protocol allows the amplification of RNA target in double stranded cDNA under standardized conditions that markedly deplete rRNA without preselecting mRNA. cDNA obtained was quality scored by RNA 6000 PicoChip for Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 1 M-NM-<g of cDNA per sample was sonicated using Covaris-S2 system (Covaris Inc., Woburn, MA, USA), cDNA fragments of 200 bp were end-repaired and adapters were ligated for HiSeq 2000 (Ilumina Inc., San Diego, CA, USA) technology using TruSeq DNA Sample kit (Illumina) and following manufacturer`s instructions. Quality of libraries was assesed using HT DNA High Sensitivity Chip (Agilent) for 2100 Bioanalyzer. Single animals per replica were euthanized with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100 M-NM-<g/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at 2,000 M-CM-^W g, 4 M-BM-0C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 M-CM-^W g to pellet insoluble material. At this point, 30 M-NM-<L of supernatant was saved as input sample. Two custom made mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The homogenate was incubated with antibody coupled beads at 4M-BM-0C with end-over-end rotation for approximately 16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 M-NM-<g/ml cycloheximide) and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) wit
2013-01-14 | E-GEOD-42880 | biostudies-arrayexpress