Affinity purification of TRMT6/61A protein complexes
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Jianwei Wang
PROVIDER: PXD047430 | iProX | Thu Nov 30 00:00:00 GMT 2023
REPOSITORIES: iProX
Publications
Age-related noncanonical TRMT6-TRMT61A signaling impairs hematopoietic stem cells.
He Hanqing H Wang Yuqian Y Zhang Xiaoting X Li Xiaoyu X Liu Chao C Yan Dingfei D Deng Haiteng H Sun Wanling W Yi Chengqi C Wang Jianwei J
Nature aging 20240117 2
Aged hematopoietic stem cells (HSCs) exhibit compromised reconstitution capacity and differentiation bias toward myeloid lineages. However, the molecular mechanism behind HSC aging remains largely unknown. In this study, we observed that RNA N<sup>1</sup>-methyladenosine-generating methyltransferase TRMT6-TRMT61A complex is increased in aged murine HSCs due to aging-declined CRL4<sup>DCAF1</sup>-mediated ubiquitination degradation signaling. Unexpectedly, no difference of tRNA N<sup>1</sup>-meth ...[more]
Similar Datasets
Project description:gDNA was estracted and digested with cocktail of restrict enzymes MseI, DdeI, AluI, MboI, incubated at 37°C for 6 h. After purified by phenol/chloroform extraction, fragmented DNA was resuspended by TE, and quantified by Qubit 3.0 (Invitrogen). For each sample, 20 μg input DNA was immunoprecipitated overnight at 4°C in a shaker, with 1× DRIP binding buffer (10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100) and 10 μg S9.6 antibody (ATCC, HB-8730). After adding 50 μl Dynabeads Protein G (Invitrogen, 10004D) and incubated for 4 h, the beads with antibody were washed by 1× DRIP binding buffer for 4 times at room temperature, each wash takes 10 min. Added 250 μl elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS) and 5 U Proteinase K to beads/antibody complexes, incubated for 40 min in an Eppendorf ThermoMixer at 55°C, 1,000 rpm. Purified by phenol/chloroform extraction, and moved supernatant to a new tube, followed by adding 1/10 volume 3 M NaAc, 1 μl GlycoBlue (Invitrogen) and 1 volume isopropanol, precipitated at -20°C for 3 h and then centrifuged. After washed by 70% ethanol and dried, the DRIPed DNA pellet was resuspended by 75 μl low EDTA TE buffer (10 mM Tris-HCl 8.0, 0.1 mM EDTA). NGS libraries were constructed using Accel-NGS® 1S Plus DNA Library Kit (Swift Biosciences).
2019-10-15 | GSE134101 | GEO
Project description:Flag-YBX1 overexpressed T24 cells pellets were resuspended with 2 volume of lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U/ml RNase inhibitor), and incubated at 4 °C for 30 min with rotation. Then the lysate was centrifuged at 15 000 g for 20 min. Before incubating the lysate with Flag beads, 100ul were taken as input. The anti-Flag M2 magnetic beads (Sigma, 10 μl per mg lysate) were washed with NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U/ml RNase inhibitor) four times. Cell lysate was mixed with M2 beads and incubated at 4 °C for 4 h with rotation. The beads were washed two times with 1 ml ice-cold NT2 buffer. Then the beads were subject to Micrococal nuclease (NEB) digestion (1:1 000 000 dilution) for 8 min at 37 °C. The beads were cooled on ice immediately for 5 min and washed two times with 1 ml ice-cold 1× PNK+EGTA buffer (50 mM Tris-HCl pH 7.5, 20 mM EDTA, 0.05% NP-40, 200 U/ml RNase inhibitor) and two times with 1 ml ice-cold 1× PK buffer (50 mM NaCl, 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.2% SDS, 200 U/ml RNase inhibitor). Then the beads were digested with 200 μl pre-heated (20 min at 50 °C) Proteinase K and RNAs were extracted with an equal volume of Acid-Phenol: Chloroform, pH 4.5 (Ambion). The RNAs were subjected to rRNA removal and Bisseq. The libraries were sequenced on the Illumina HiSeq X-Ten platform at Novogene (Tianjin, CA) with paired-end 150 bp read length.The m5C sites were called using meRanCall from meRanTK (FDR < 0.01).
2019-07-02 | GSE133625 | GEO
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.
2021-09-05 | GSE152862 | GEO
Project description:Purpose: this study is to analyze the chromatin landscape of H3R2mes in Plasmodium falciparum. Methods: In this study, H3R2me2s enrichment in the chromatin of wildtype parasite at schizont stage was analyzed by Cleavage Under Targets and Tagmentation (CUT&Tag) coupled with next generation sequencing (CUT&Tag-seq). Synchronized WT 3D7 at 40–46 hpi schizonts were harvested and fixed with 1% formaldehyde for 1 min at room temperature, followed by lysis with 0.06% saponin. The parasite pellets were suspended in 100 μl nuclear extraction buffer (20 mM HEPES–KOH pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5 mM Spermidine, 1x EDTA-free Protease Inhibitor cocktail). Ten μl of activated Concanavalin A-coated magnetic beads (EpiCypher # 21-1401) were added to each sample (~0.5 × 106 nuclei) and incubated for 10 min. The bead-bound nuclei were resuspended in 50 μl Antibody150 buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitor cocktail, 0.01% digitonin, 2 mM EDTA) containing 0.5 μg of rabbit anti-H3R2me2s (Epigentek #A-3705-050) as the primary antibody or rabbit IgG (Cell Signaling Technology #2729S) serving as a control. The primary antibody was then removed, and nuclei were then incubated with 0.5 μg of the secondary antibodies in 50 μl of Digitonin150 buffer (Antibody150, no EDTA) at room temperature for 30 min. The secondary antibodies were mouse anti-rabbit IgG (Invitrogen #31213). The nuclei were washed thrice with 200 μl Digitonin150 Buffer for 5 min to remove unbound antibodies and finally resuspended in 50 μl Digitonin300 Buffer (Digitonin150 but with 300 mM NaCl). Then, 2.5 μl of CUTANA pAG-Tn5 (EpiCypher #15-1017) were added to each reaction at room temperature for 1 h. The nuclei were washed thrice for 5 min in 200 μl Digitonin300 Buffer to remove the unbound pA-Tn5. Next, the nuclei were resuspended in 50 μl Tagmentation buffer (10 mM MgCl2 in Digitonin300) and incubated at 37 °C for 1 h. The beads were washed with 50 µl TAPS buffer (10 mM TAPS, pH 8.5, 0.2 mM EDTA), mixed with 5 µL SDS release buffer (10 mM TAPS pH 8.5; 0.1% SDS) to quench tagmentation, and then incubated for 1 hr at 58ºC to release tagmented chromatin into solution. Finally, 15 µl of 0.67% Triton-X was added to each reaction to quench SDS. PCR with appropriate barcoded primers was performed using the CUTANA High Fidelity 2x PCR Master Mix (EpiCypher #15-1018) according to the manufacturer's recommendations. Amplified DNA libraries were captured by incubating with 1.3 × Kapa pure beads (Roche #KK8001) according to the manufacturer's recommendations, and next-generation sequencing was performed on the NextSeq 550 platform. The reads of CUT&Tag-seq from three biological replicates were mapped to the P. falciparum genome (PlasmoDB Release 39.0) by using BWA (Li and Durbin 2009) after FastQC quality control. The peak calling was finished by MACS2 version 2.2.7.1 with parameters ‘callpeak -f BAMPE’ and a q-value cutoff of 0.05. Results: The three replicates of CUT&Tag-seq using nuclei from the schizont stage showed high reproducibility, with correlation coefficients of ~0.8. Peak calling using criteria of presence in at least two of three replicates with at least 50% overlap of the peaks identified 1629 H3R2me2s signals, distributed among the 14 chromosomes. Among these peaks, 57% (927) are localized at 5’ UTR regions of 791 genes, 35% (577) at coding regions of 470 genes, and 8% (125) at 3’ UTR regions of 115 genes. The peaks are ~17.44 bp wide and ~1265 bp from the ATG sites. Comparing the transcriptome data showed genes with H3R2me2s enrichment in the 5’ UTRs showed significantly higher expression than other genes in the genome, suggesting that H3R2me2s is associated with gene activation. In addition, H3R2me2s peaks were highly co-localized with the active mark H3K9Ac, H2A.Z, and H3K4me3, further implying H3R2me2s as a euchromatin mark. Gene ontology (GO) enrichment analysis revealed a multitude of cellular pathways, including merozoite invasion, transcription, translation, stress response, cell cycle, schizogony, cell adhesion, exit from RBC, vesicle transport, signal transduction, DNA repair, and telomere organization, which are potentially regulated by this euchromatic mark. Conclusions: Collectively, H3R2me2s is a active chromatin mark and regulates invasion and other important processes in human malaria parasite.
2023-06-28 | GSE214535 | GEO
Project description:Ten 15 cm2 plates of HEK293 cells stably expressing TAP-vector, TAP-CSAG1, or TAP-CSAG2 were lysed with TAP lysis buffer (10% (v/v) glycerol, 50 mM HEPES-KOH pH 7.5, 100 mM KCl, 2 mM EDTA, 0.1% (v/v) NP-40, 10 mM NaF, 0.25 mM Na3VO4, 50 mM β-glyerolphosphate, 2 mM DTT, and 1X protease inhibitor cocktail (Sigma)) and cleared supernatants were bound to IgG-Sepharose beads (GE Amersham) and then washed in lysis buffer and TEV buffer (10 mM HEPES-KOH pH 8.0, 150 mM NaCl, 0.1% (v/v) NP-40, 0.5 mM EDTA, 1 mM DTT, and 1X protease inhibitor cocktail). Protein complexes were cleaved off the beads by TEV protease (Sigma) and incubated with calmodulin-Sepharose beads (GE Amersham) in calmodulin binding buffer (10 mM HEPES-KOH pH 8.0, 150 mM NaCl, 1 mM Mg acetate, 1 mM imidazole, 0.1% (v/v) NP-40, 6 mM CaCl2, 10 mM 2-mercaptoethanol) and then washed in calmodulin rinse buffer (50 mM Ammonium bicarbonate pH 8.0, 75 mM NaCl, 1 mM Mg Acetate, 1 mM Imidazole, 2 mM CaCl2) before eluted with SDS sample buffer, subjected to SDS-PAGE, and stained with GelCode Blue stain (Thermo Fisher Scientific) before protein identification by LC-MS/MS.
2020-09-01 | PXD020231 | Pride
Project description:J1 mouse embryonic stem cells (mESCs) and NIH-3T3 fibroblasts were grown in standard media conditions. Hybridized cells were fixed with 4% paraformaldehyde; permeabilized in 70% ethanol; incubated for 12 hours at 30C in an RNA preserving hybridization (RPH) buffer (300 mM Sodium chloride, 30mM Sodium citrate, 2.1M Ammonium sulfate, 25% formamide, 10 mM EDTA, 1 mg/ml E. Coli tRNA, 500 μg/ml BSA); and reverse cross-linked for 1 hour at 50C with Sodium dodecyl sulfate (SDS) and Proteinase K (100 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.5% SDS, 500 μg/ml Proteinase K).
2014-03-20 | GSE55919 | GEO
Project description:In order to identify the molecular mechanism of MCPH1 antagonizing necroptosis， we performed affinity purification of MCPH1 protein complexes in TSZ-treated L929 cells, L929 cells were stably expressing Flag-tagged MCPH1 and treated with T+S+Z (T: 50 ng/ml TNFα, S: 100 nM SmacM, Z: 20 μM zVAD) for 1 hour. The resulting cells were lysed in NETN buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40, containing protease and phosphatase inhibitors cocktail) on ice for 30 min. Cell debris were eliminated by centrifugation and the supernatant was incubated with anti-Flag beads. After the 4 hours incubation, beads were washed with 1 mL NTEN buffer for three times, beads were boiled in 30 μL 2×SDS loading buffer and the immunocomplexes were eluted. Samples were resolved on 10% SDS-PAGE and analyzed by mass spectrometry. To elucidate the molecular mechanism behind MCPH1 nuclear translocation upon DNA damage, we performed affinity purification of MCPH1 protein complexes in response to camptothecin (CPT) treatment. HEK293T cells stably expressing SFB-tagged MCPH1 were lysed in NETN buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40, containing protease and phosphatase inhibitors cocktail) on ice for 30 min. Cell debris were eliminated by centrifugation and the supernatant were obtained and incubated with 50 μL S-protein beads at 4 ℃. After the 4 hours incubation, beads were washed with 1 mL NTEN buffer for three times, beads were boiled in 30 μL 2×SDS loading buffer and the immunocomplexes were eluted. Samples were resolved on 10% SDS-PAGE and analyzed by mass spectrometry.
2024-01-13 | PXD048580 | iProX
Project description:Mouse induced pluripotent stem cells (iPSCs) were derived from embryonic fibroblasts by overexpressing the Yamanaka factors Oct4, Sox2, Klf4 and c-Myc, and then grown in standard 2i/serum media conditions. Hybridized iPSCs were fixed with 4% paraformaldehyde; permeabilized in 70% ethanol for over 12 hours; incubated for 12 hours at 30C in an RNA preserving hybridization buffer (300 mM Sodium chloride, 30mM Sodium citrate, 2.1M Ammonium sulfate, 25% formamide, 10 mM EDTA, 1 mg/ml E. Coli tRNA, 500 μg/ml BSA); and reverse cross-linked for 1 hour at 50C with Sodium dodecyl sulfate (SDS) and Proteinase K (100 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.5% SDS, 500 μg/ml Proteinase K).
2014-03-20 | GSE55672 | GEO
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 37°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 4°C, the cell pellets were resuspended in 100 μl ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4°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 4°C). 100 μl 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 4°C overnight with 6 μg/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 μl pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4°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 μl 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 65°C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (QIAGEN) and eluted into 30 μl H2O according to the manufacturerâs protocol after treatment with RNase A and Proteinase K.
2016-07-24 | E-GEOD-77854 | biostudies-arrayexpress
Project description:Chromatin immunoprecipitation was performed with modification of the protocols described before (Lee et al., 2006; Lilja et al., 2007). Briefly, the chorion was removed from 12-hour old embryos, cross-linked using 2% paraformaldehyde and resuspended in storage buffer (50mM Tris-HCl pH8.0, 1 mM EDTA). Embryos were then lysed in SDS-lysis buffer (1.0 ml 5 M NaCl, 2.5 ml 1 M Tris-HCl pH 8.0, 0.5 ml 0.5M EDTA, 2.5 ml 10 % SDS), resuspended in SDS-lysis and Triton buffer (1.0 ml 5 M NaCl, 5.0 ml 1 M Tris-HCl pH 8.0, 0.5 ml 0.5M EDTA, 2.5 ml Triton X-100, protease inhibitor cocktail) and sonicated on ice to an average length of 350 bp. The resulting sheared chromatin (25Izg) was subjected to immunoprecipitation using anti-Myc antibody (Santa Cruz) as described previously (Lee et al., 2006). ChIP-sequencing libraries were constructed following manufactureras instructions (Illumina). The resulting DNA libraries were sequenced using Illumina platform (University of Massachusetts Medical School Core Facility).
2019-02-02 | E-MTAB-4083 | biostudies-arrayexpress