Dataset Information

MB135-iDUX4ca HSATII RNP ChIRP LC/MS

ABSTRACT: ChIRP was performed as previously described (Chu et al., 2012) with modifications. 6-10 15 cm plates of cells were used per ChIRP-MS experiment. Cells are cross-linked in 2% paraformaldehyde for 15 minutes at RT, followed by 0.125 M glycine quenching for 5 minutes, scraped and pellet at 650xg for 5 minutes at 4C. Cells were then lysed in lysis buffer (50 mM Tris-HCl, pH 7.0, 10 mM EDTA, pH 8.0, 1% SDS supplemented with fresh 0.5 mM AEBSF (ThermoFisher Scientific, cat# BP635-500), Pierce Protease Inhibitors EDTA-free (PIA32955) and Pierce Phosphatase Inhibitors (PIA32957) and RNase inhibitor (TaKaRa, cat# 2313A)) for 10 minutes on ice. Samples were sonicated in a Biorupter (Diagenode) for 15 minutes on high, 30 sec on/ 30 sec off pulse intervals for 4-cycles. Samples were centrifuged at 16,000xg for 10 minutes at 4C and 5% of the lysate was taken out as an input control. Lysates were pre-cleared with beads (Invitrogen Dynabeads M-280 Streptavidin, cat# 11205D) at 37C for 30 minutes at 800rpm. Hybridization was performed at 37C for 4 hours shaking at 800rpm using biotin-conjugated probes (HSATII probe: 5`-ATTCCATTCAGATTCCATTCGATC-3BioTEG-3`, Control probe: 5`-GTCCCGTTAGCTCAGGTGGTAGAGCAC-3BioTEG-3` or 5`-TGCTGATGAAGCAGAACAAC-3BioTEG-3`) in hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 15% formamide (Sigma-Aldrich) and supplemented with fresh 0.5 mM AEBSF (ThermoFisher Scientific, cat# BP635-500), Pierce Protease Inhibitors EDTA-free (PIA32955) and Pierce Phosphatase Inhibitors (PIA32957) and RNase inhibitor (TaKaRa, cat# 2313A)). Streptavidin magnetic beads (Invitrogen Dynabeads M-280 Streptavidin, cat# 11205D) were added (100 uL per 100 pmole of probes used) and incubated at 37C for 30 minutes at 800rpm. Beads were washed 3x in pre-warmed (37C) wash buffer (2X SSC (Invitrogen), 0.5% SDS and supplemented with Pierce Protease Inhibitors EDTA-free (PIA32955) and Pierce Phosphatase Inhibitors (PIA32957) and RNase inhibitor (TaKaRa, cat# 2313A) and 1 mM DTT) for 5 minutes at 37C at 800rpm. 10% of beads were removed for RNA isolation and 90% of remaining beads were used for protein isolation. RNA was isolated in RNA elution buffer (10 mM EDTA, pH 8.0, 95% formamide (ThermoFisher Scientific)) and incubated at 95C for 5 minutes at 1,000xg. Proteinase K was added (20mg/mL, ThermoFisher Scientific, cat# AM2546) and incubated at 55C for 1 hour. RNA was extracted using TRIzol and validation of HSATII enrichment was performed using RT-qPCR. Protein was isolated by boiling beads for 30 minutes at 95C in 2X NuPage LDS sample buffer (ThermoFisher Scientific, cat#NP0007). Protein samples were using in immunoblotting or for LC-MS. For LC-MS, eluted protein samples were electrophoresed into a NuPage 4-12% Bis-Tris gel, excised, and processed by the Fred Hutchinson Cancer Research Center Proteomics Core. Samples were reduced, alkylated, digested with trypsin, desalted, and run on the Orbitrap Eclipse Tribid Mass Spectrometer (ThermoFisher Scientific). Proteomics data were analyzed using Proteome Discoverer 2.4 against a UniProt human database that included common contaminants using Sequest HT and Percolator for scoring. Results were filtered to only include protein identifications from high-confidence peptides with a 1% false discovery rate. Proteins that were identified in all replicates from both independent experiments with at greater than ten unique peptide matches and greater than 1.5 difference between sample and control were analyzed further.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Stephen J. Tapscott

PROVIDER: MSV000096938 | MassIVE | Fri Jan 24 15:15:00 GMT 2025

SECONDARY ACCESSION(S): PXD060172

REPOSITORIES: MassIVE

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:RICK was performed as previously described (Bao et al., 2018). Briefly, cells were cultured in 15cm plates and 0.1mM 5-ethynyl uridine (ThermoFisher Scientific) was incubated with cells after dox-induction for 16-hrs followed by a washout and then fixed at 48-hrs. After washing 3x with 1X PBS, the plates were placed on ice and irradiated with 0.15 J/cm2 UV light at 254 nm (Stratalinker). Cells were then fixed with 90% ethanol for 30 min, washed 3x with 1X PBS, and permeabilized with 0.5% Triton X-100 in 1X PBS for 15 min. Permeabilized cells were incubated with 15 mL of click reaction buffer (0.25M biotin-azide (Click Chemistry Tools, cat# 1265-25), 0.5M CuSO4, 0.25M THPTA, 0.1M sodium L-ascorbate in 1X PBS) for 30 minutes at RT. Reaction was quenched 3x in 0.5% Triton X-100 in 1X PBS with 2mM EDTA for 3 minutes at RT. Cells were then lysed in lysis buffer (20 mM Tris-HCl, pH 7.5, 500 mM LiCl, 1 mM EDTA pH 8.0, 0.5% lithium-dodecylsulfate (LiDS), and 5 mM DTT) supplemented with Pierce Protease Inhibitors EDTA-free (PIA32955) and Pierce Phosphatase Inhibitors (PIA32957) and RNase inhibitor (TaKaRa, cat# 2313A), and harvested by scraping. Samples were sonicated in a Biorupter (Diagenode) for 15 min on low, 30 sec on/ 30 sec off to aid in lysis. Samples were centrifuged at 12,000 rcf for 10 min at 4C and 5% of the lysate was taken out as an input control. Complexes containing different RNA species, and their associated proteins, were isolated with streptavidin-conjugated magnetic beads (100 ul beads for each plate; Dynabeads MyOne Streptavidin C1, Thermo Fisher Scientific, cat# 65602). After incubation with the lysates overnight under continuous rotation at 4C, the beads were isolated on a magnetic stand and washed using lysis buffer, buffer 1 (20 mM Tris-HCl, pH 7.5, 500 mM LiCl, 1 mM EDTA pH 8.0, 0.1% LiDS, and 5 mM DTT), buffer 2 (20 mM Tris-HCl pH 7.5, 500 mM LiCl, 1 mM EDTA pH 8.0, and 5 mM DTT), and buffer 3 (20 mM Tris-HCl pH 7.5, 200 mM LiCl, 1 mM EDTA pH 8.0, and 5 mM DTT) for two times each under rotation (for 10 minutes at 4C). At last wash step beads were split to isolate RNA or proteins. Proteins were extracted from the captured complexes using RNase A/T1 mix (ThermoFisher Scientific, cat# EN0551) and Ambion RNase III (ThermoFisher Scientific, cat# AM2290) at 37C for 1 hour at 1,000xg, then boiled for 10 minutes 95C. RNA was isolated in RNA elution buffer (10 mM EDTA, pH 8.0, 95% formamide (ThermoFisher Scientific)) and incubated at 95C for 5 minutes at 1,000xg. Proteinase K was added (20mg/mL, ThermoFisher Scientific, cat# AM2546) and incubated at 55C for 1 hour. RNA was extracted using TRIzol. For LC-MS, eluted protein samples were electrophoresed into a NuPage 4-12% Bis-Tris gel, excised, and processed by the Fred Hutchinson Cancer Research Center Proteomics Core. Samples were reduced, alkylated, digested with trypsin, desalted, and run on the Orbitrap Eclipse Tribid Mass Spectrometer (ThermoFisher Scientific). Proteomics data were analyzed using Proteome Discoverer 2.4 against a UniProt human database that included common contaminants using Sequest HT and Percolator for scoring. Results were filtered to only include protein identifications from high-confidence peptides with a 1% false discovery rate. Proteins that were identified in all replicates from both independent experiments with at least two unique peptide matches and greater than 1.5 difference between sample and control were analyzed further.

Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.

Project description:U2OS osteosarcoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) and Raji B-cells in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS, Bio&Sell), 2 mM L-glutamine (Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco) at 37°C at 8% or 5% CO2, respectively. Chromatin immunoprecipitation for ChIP-seq: Cells were crosslinked using a formaldehyde containing solution (10mM NaCl, 0,1mM EDTA pH 8, 0,05mM EGTA pH 8, 5mM HEPES pH 7.8 and 1% formaldehyde) for 10 minutes at 20°C, the reaction was quenched by the addition of glycin to a final concentration of 250uM for 5 minutes. Crosslinked cells were collected and washed twice with PBS before snap freezing in liquid nitrogen and storage at -80°C until subsequent use. Prior to sonication, the crosslinked cells were resuspended in lysis buffer (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA pH 8, 10% glycerol, 0.75% NP-40, 0.25% Triton X-100, 1X protease inhibitor cocktail) at 4°C for 20 minutes. Nuclei were collected by centrifugation and washed in a second buffer (200mM NaCl, 1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 1X protease inhibitor cocktail) for 10 minutes at 4°C then collected by centrifugation and resuspended in the shearing buffer (1mM EDTA pH 8, 0.5mM EGTA pH 8, 10mM Tris pH 8, 100mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1X protease inhibitor cocktail). Sonication was carried out in a Bioruptor Pico ultrasounds water bath (Diagenode B01060001) for 30 cycles of 30 sec ON and 30 sec OFF pulses in 4°C water. Sonicated extracts were centrifuged at high speed in the presence of 0,1% of Triton X-100 and snap frozen in liquid nitrogen then stored at -80°C until subsequent use. Prior to ChIP, the ZNF768 mAb was coupled to protein-G coated magnetic beads (Dynabeads, life technologies) by incubation in 0,5% BSA PBS overnight at 4°C. Pre-coated beads were then washed and incubated with the sonicated chromatin extracts, the ChIP was carried out overnight at 4°C on a rotating wheel. The equivalent of 10 × 107 cells sonicated extract was used for each ZNF ChIP experiment for both cell lines. After incubation, the beads were washed 7 times with Wash buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA pH 8, 1% NP-40, 0.7% Na-Deoxycholate, 1X protease inhibitor cocktail) followed by one wash with TE-NaCl buffer (10mM Tris pH 8 and 1mM EDTA pH 8, 50mM NaCl). Immunoprecipitated chromatine was eluted by two sequential incubations with 100µl Elution buffer (50mM Tris pH 8, 10mM EDTA pH 8, 1% SDS) at 65°C for 15 minutes. The two eluates were pooled and incubated at 65°C for 12 hours to reverse-crosslink the chromatin followed by treatment with RNase A (0.2µg/mL) at 37°C for two hours and Proteinase K (0.2µg/mL) at 55°C for two hours. The DNA was isolated by phenol:chloroform: isoamylalcohol (25:24:1 pH 8) extranction followed by Qiaquick PCR Purification (Qiagen, Germany) and quantified with Qubit DS DNA HS Assay (ThermoFisher Scientific, USA). At least 1ng of ChIP DNA was used to prepare sequencing library with Illumina ChIP Sample Library Prep Kit (Illumina, USA) with a few optimizations to the protocol. The ChIP DNA was size selected using Ampure beads (Life technologies) to enrich for fragments < 400Bp prior to end-repair, 3’end adenylation and adapter ligation. Library fragments were then directly amplified by 10 cycles of PCR.

Project description:Identification and characterization of HP1BP3 (a human histone H1 homologue) as a novel chromatin retention factor essential for the co-transcriptional processing of pri-miRNA. We generated BAC transgenic cells at 80% confluency (~1x107) were cross-linked with 1% formaldehyde for 10 minutes at 37Â°C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4Â°C, the cell pellets were resuspended in 100 Î¼l ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4Â°C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 15 minutes). The sheared chromatin with a fragment length of ~200 â 600 bp) was centrifuged at 20,000 g for 15 minutes at 4Â°C). 100 Î¼l of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4Â°C overnight with 6 Î¼g/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitation was carried out by incubating with 40 Î¼l pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4Â°C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 Î¼l ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65Â°C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (QIAGEN) and eluted into 30 Î¼l H2O according to the manufacturerâs protocol after treatment with RNase A and Proteinase K.

Dataset Information

MB135-iDUX4ca HSATII RNP ChIRP LC/MS

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets