Dataset Information

Identification of spidroin proteins in the silk glands of Nephila clavipes spiders

ABSTRACT: Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Trichonephila Clavipes

TISSUE(S): Silk Gland

SUBMITTER: Inmaculada Jorge

LAB HEAD: Jesús Vázquez Cobos

PROVIDER: PXD023141 | Pride | 2022-12-07

REPOSITORIES: Pride

ACCESS DATA

Similar Datasets

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: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.

Project description:Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long non-coding RNAs (lncRNAs) recruit PRC2 to chromatin, but the general role of RNA in maintaining repressed chromatin is unknown. ChIP-seq, combined with RNA-seq, indicating that PRC2 is also associated with active genes, but most of these are not regulated by PRC2. These results were complemented by in vitro binding assays measuring the binding constant of human PRC2 to various RNAs and find comparable affinity for human lncRNAs targeted by PRC2 and irrelevant transcripts from ciliates and bacteria. PRC2 binding is size-dependent, with lower affinity for shorter RNAs. These findings support a model in which promiscuous binding of PRC2 to RNA transcripts allows it to scan for target genes that have escaped repression, leading to maintenance of the repressed state. Such RNAs may also provide a decoy for PRC2. Cell culture: HEK293T/17 cells were cultured in DMEM with 10% FBS for no longer than 15 passages. ON-TARGETplus SMARTpool for human SUZ12 (Thermo Scientific, Dharmacon cat # L-006957-00-0005) was used to knockdown SUZ12 (siSUZ12) and ON-TARGETplus Non-targeting Pool (Dharmacon cat # D-001810-10-05) was used as a negative control (siCtrl). A total of 25 nM siRNA was transfected in 6-well dishes using LipofectamineM-bM-^DM-" RNAiMAX Reagent (Life Technologies, Invitrogen) following the manufacturerM-bM-^@M-^Ys recommendations. RNA-seq: Polyadenylated RNA was purified from 4 ug of RNA. cDNA libraries were prepared and double-stranded cDNA was fragmented using DNase I according to Illumina specifications, prior to adaptor ligation. Sequencing libraries were amplified and sequenced using an Illumina HiSeq 2000 sequencer. ChIP-seq: 30 million cells at 80% to 90% confluent culture were crosslinked in 1% v/v formaldehyde for 10 min, quenched in 150 mM glycine, washed with cold 1xPBS and harvested by scraping. Cells were lysed in Lysis Buffer (1% w/v SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) with 1x CompleteM-BM-. protease inhibitors (Roche). Cells were sonicated for 10 to 15 min using a BioruptorM-bM-^DM-" UCD-200 (Diagenode) with 30 sec pulses at maximum power. Lysates were diluted to 10 ml in IP Buffer (0.01% w/v SDS, 1.1% v/v Triton-X, 1.2 mM EDTA, 16.7 mM Tris-HCL pH 8.0, 167 mM NaCl) and 10 to 20 ng of antibodies were added and incubated overnight at 4 M-bM-^AM-0C with rotation. Antibodies were immunoprecipitated with 60 M-BM-5l protein G Plus/protein A Agarose Suspension (Calbiochem cat # IP05) and washed sequentially with 1 ml Low Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), 1 ml High Salt Wash Buffer (0.1% w/v SDS, 1% v/v Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), 1 ml LiCl Wash Buffer (0.25 M LiCl, 1% v/v Nonidet P-40, 1% w/v deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0) and 1 ml TE buffer (Qiagen). DNA was eluted with 0.4 ml of 0.1 M NaHCO3 and 1% w/v SDS. Eluent was transferred into a new tube and NaCl was added to a final concentration of 200 mM. Crosslinks were reversed by incubation at 65 M-bM-^AM-0C for 2 hours. Next, proteins and RNA were digested by adding 33 M-BM-5l of Digestion Reagent (0.6 M Tris-HCl pH 6.5, 152 mM EDTA, 61 ng/ml RNase A (Invitrogen cat # AM2274) and 0.61 mg/ml Proteinase K (NEB cat # P8102)) following by 1 hour incubation at 37 M-bM-^AM-0C. DNA was extracted by phenol:chloroform and ethanol precipitated. DNA was resuspended in Milli-Q pure water and concentration was measured using QubitM-bM-^DM-" (Invitrogen). At least 10 ng of recovered DNA was used to synthesize sequencing libraries using the ChIP-seq Sample Preparation kit (Illumina). Between 6 and 10 pmoles were used for sequencing on the HiSeq2000 sequencer.

Dataset Information

Identification of spidroin proteins in the silk glands of Nephila clavipes spiders

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure