OEX_Zengwei_2208181103
Ontology highlight
ABSTRACT:
TISSUE(S): ['human Intrahepatic Cholangiocarcarcinoma Cell Line']
SUBMITTER: Zengwei Tang
PROVIDER: OEX00020408 | NODE | 2022-08-18
REPOSITORIES: NODE
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Project description:Huh7.5 cells were transfected with HCV-JFH1 RNAand harvested after 48h of transfection. Total proteins were extracted in IP lysis buffer (Pierce, Thermo Scientific) containing 1× halt-protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA) with intermediate vortexing followed by mild sonication. The lysate was centrifuged at 14,000 × g for 30 min at 4 °C. The supernatant was quantified by the BCA method (Thermo Fisher Scientific, USA). An equivalent amount (4 mg) of proteins from each condition was incubated with 3g of anti-HuR antibody for overnight at 4 °C. The immunocomplex was captured by using protein G Sepharose 4 fast flow beads (17-0618-01/ GE Healthcare). The immunoprecipitated complex was washed two times with IP lysis buffer and one time with mili-Q. Bound protein complexes were eluted in 50 µl SDS-PAGE 2X Laemmli sample buffer. The samples were resolved on 12% SDS–PAGE and stained with Coomassie stain (0.1% w/v). The gel was subjected to further in-gel mass spectrometry analysis.
2023-09-16 | PXD035903 | Pride
Project description:The cells were cotransfected with epitope-tagged expression plasmids or empty vector(EV) . Forty-eight hours after transfection, the cells were harvested with 500 μl of lysis buffer (20 mM Tris-HCl, pH=8.0; 100 mM NaCl; 0.5% NP-40; and 1 mM EDTA) supplemented with protease inhibitor cocktail (#04693132001, Roche) and PMSF (#PB0425). The cell lysate was rotated at 4°C for 30 min. After centrifugation at 13 000 × g for 10 min, the supernatants were used for immunoprecipitation (IP). The cell extracts were incubated with anti-Flag antibody (F1804 Sigma) overnight at 4°C, followed by incubation with lysis buffer and washing with 50 μL of protein G agarose resin (Yeasen, 36405ES08) for 3 h. The incubated protein G was washed with lysis buffer at 4°C for 10 min. The cell extracts (50 μL of input) and IP samples were resuspended in 80 μL of SDS buffer, followed by pipetting up and down several times to mix the samples.For mass spectrometry analysis of heterochromatin-associated proteins and murine Nat10-associated proteins, the immunoprecipitate was subjected to 10% Acr-Bis SDS‒PAGE and separated approximately 2 cm from the well. The gel was cut and used for mass spectrometry analysis by the PTM BIO Company (Hangzhou, China).
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Project description:To analyze the primary STAT3 binding sites induced by OSM, chromatin immunoprecipitation sequencing (ChIP-seq) was performed using an anti-human STAT3 antibody. HHOs were cultured in EM for 1 week and cultured EM (-OSM) for 24 hrs to deplete OSM before OSM induction, and then incubated with EM(+OSM) for 0.5 hr. HHOs at 0h and 0.5h after OSM induction were subjected to ChIP-seq analysis using ChIPmentation methodology with minor adaptations [Schmidl et al. PMID: 26280331]. Briefly, organoids were dissociated into single cells and fixed for 10 min, and then quenched by glycine. The lysed and sonicated chromatin was transferred to a new tube. The chromatin was reacted with the anti-STAT3 antibody (Cell Signaling Technology: Stat3 (D3Z2G) Rabbit mAb #12640) on a rotator overnight at 4°C. Washed and blocked Dynabeads Protein A/Protein G magnetic beads in RIPA-LS supplemented with 0.1% BSA were added to the lysates and incubated for 2 hr on a rotator at 4°C. Beads were subsequently washed with RIPA-LS, RIPA-HS , RIPA-LiCl, and Tris-Cl pH 8.0. The beads were then resuspended in a tagmentation reaction mix containing 1 μl of TDE1 Tagment DNA Enzyme (Illumina) and incubated at 37°C for 3 min on a thermocycler. The beads were further washed and incubated with Pronase for 1 hr at 42°C and 8 hr at 65°C to revert cross-linking. Finally, DNA was purified with MinElute columns and eluted with elution buffer. The DNA was subsequently amplified with Nextera sequencing primers and NEBNext high fidelity 2x PCR master mix for 5–10 cycles. Enriched libraries were purified, size selected using AMPure XP beads to recover libraries with the fragment length of 200–400 bp, and sequenced by llumina HiSeq 2500 sequencer with 150 bp paired-end reads.
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Project description:The CLIP procedures were conducted using HeLa cells according to the previous study (Liu et al., 2016) with slight modifications. Briefly, 4 plates of HeLa cells in 15 cm dishes were transfected with pPB/ALKBH3 plasmid for 48 h to reach about 90% cell confluency. After washing twice with ice-cold PBS, cells were irradiated twice with 400 mJ/cm2 at 254 nm by Stratalinker on ice. Cells were lysed in high salt lysis buffer (300 mM NaCl, 0.2% NP-40, 20 mM Tris-HCl PH 7.6, 0.5 mM DTT, protease inhibitor cocktail (1 tablet/50 ml), and RNase inhibitor (1:200)) at 4°C for 30 min. Supernatant was collected after centrifuged at 17,000 g at 4°C for 15 min and further treated with 1 U RNase T1 for 15 min at 24 °C. After centrifugation and filtration through 0.22 µM filter, 10% supernatant was collected as input for further sequencing analysis. Anti-Flag M2 beads (Sigma-Aldrich, St. Louis, MO, 80 μl) were washed three times with lysis buffer and incubated with the filtered supernatant at 4°C for 4 hrs. After removing the flow through by magnetic rack, beads and input were further incubated with 10 U RNase T1 exactly for 8 min. Samples were treated with 95 µL commercial PNK buffer and 0.5 U PNK for 15 min at 37 °C. After treatments, final concentration of 100 µM ATP and 1 U PNK were added and incubated at 37 °C for another 30 min. The RNA/protein complexes were eluted by NuPAGE 1 × loading buffer and fractionated by neutral NuPAGE 4–12% bis-tris gel. After cutting the gel, RNAs were recovered by proteinase K digestion in proteinase K buffer, followed by phenol/chloroform extraction using glycogen as carrier. The RNA fragments were ligated to adapter and established library by NEB small RNA library preparation kit (E7330S).
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