LitCovid-sentences  

PMC:7640975 / 9881-12865 JSONTXT 7 Projects

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Id Subject Object Predicate Lexical cue
T50 0-54 Sentence denotes 2.5 Protein Extraction, Digestion, and iTRAQ Labeling
T51 55-259 Sentence denotes At 24 h postinfection (hpi), both PDCoV- and mock-infected IPEC-J2 cells grown in T25 flasks (∼5 × 106 cells/flask) were rinsed twice with prechilled PBS, and then harvested with disposable cell scrapers.
T52 260-370 Sentence denotes Three flasks of each group (PDCoV or mock) were harvested and used as three independent biological replicates.
T53 371-580 Sentence denotes After centrifugation at 300g for 10 min, the cell pellets from each flask were lysed with 800 μL of RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% SDS, 8 M urea and protease inhibitors (Beyotime).
T54 581-686 Sentence denotes The cell lysates were further sonicated on ice for 5 min with 10 s bursts and 10 s pauses between cycles.
T55 687-810 Sentence denotes After centrifugation for 20 min at 12 000g and 4 °C, the supernatant was collected and used as the total cellular proteins.
T56 811-939 Sentence denotes The concentration of protein samples was determined using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA).
T57 940-1121 Sentence denotes The total cellular proteins from each biological replicate were equally divided into three aliquots, which were used as three independent technical replicates for the LC-MS/MS runs.
T58 1122-1310 Sentence denotes For tryptic digestion and iTRAQ labeling, an aliquot of each protein sample containing ∼100 μg of total cellular proteins was adjusted to a 100-μL final volume using the RIPA lysis buffer.
T59 1311-1460 Sentence denotes A final concentration of 10 mM of tris (2-carboxyethyl) phosphine (TCEP) was added to each protein sample, which was then incubated at 37 °C for 1 h.
T60 1461-1621 Sentence denotes Afterward, iodoacetamide was added at a final concentration of 40 mM, and the protein solution was incubated for 40 min at room temperature shielded from light.
T61 1622-1742 Sentence denotes Subsequently, prechilled acetone was added to the protein solution in a ratio of 6:1 and precipitated at −20 °C for 4 h.
T62 1743-1880 Sentence denotes After centrifugation (10 000g, 4 °C) for 20 min, the precipitate was dissolved with 100 μL of 100 mM triethylammonium bicarbonate (TEAB).
T63 1881-1968 Sentence denotes The processed protein samples were digested with 2 μg/μL of trypsin overnight at 37 °C.
T64 1969-2099 Sentence denotes Following tryptic digestion, the generated peptides were dried by vacuum centrifugation, and redissolved with 20 μL of 0.5 M TEAB.
T65 2100-2242 Sentence denotes The prepared peptides were then labeled with an iTRAQ reagents-8 plex kit (AB Sciex, Foster City, CA, USA) as per the manufacturer’s protocol.
T66 2243-2519 Sentence denotes Briefly, the three independent biological replicates of mock-infected cellular samples were each labeled with iTRAQ-113, iTRAQ-114, and iTRAQ-115; the three independent biological replicates of PDCoV-infected samples were each labeled with iTRAQ-116, iTRAQ-119, and iTRAQ-121.
T67 2520-2795 Sentence denotes All the labeled samples of each group were mixed with an equal amount, and then fractionated using an ACQUITY ultra performance liquid chromatography (UPLC) system (Waters Corp., Milford, MA, USA) combined with an ACQUITY UPLC BEH C18 column (300 Å, 1.7 μm, 2.1 mm × 150 mm).
T68 2796-2858 Sentence denotes Finally, a total of 10 fractions of each group were collected.
T69 2859-2984 Sentence denotes After merging two fractions of each group into one, the pooled 10 fractions were dried by using a rotary vacuum concentrator.