T. pui samples were collected in the Tibetan Plateau Peculiar Bio-resources Research Station of Sun Yat-sen University at Nyingchi in the Tibet Autonomous Region (4,156 m altitude, 29°36′N, 94°36′E) from June to August in 2014. The larvae were reared as previously described (Sun et al. 2011 (link), Wu et al. 2015 (link)). The larvae of different instars (third through eighth), female pupae of different ages (1, 9, 17, and 33 d old), and 1-d-old adult females were used for the analysis of gene expression. All of the samples at different developmental stages were freshly frozen in a Sample Protector (Takara, Tokyo, Japan) at −80°C until RNA isolation.
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7 protocols using sample protector
1
Transcriptome Analysis of Tibetan Pui
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T. pui samples were collected in the Tibetan Plateau Peculiar Bio-resources Research Station of Sun Yat-sen University at Nyingchi in the Tibet Autonomous Region (4,156 m altitude, 29°36′N, 94°36′E) from June to August in 2014. The larvae were reared as previously described (Sun et al. 2011 (link), Wu et al. 2015 (link)). The larvae of different instars (third through eighth), female pupae of different ages (1, 9, 17, and 33 d old), and 1-d-old adult females were used for the analysis of gene expression. All of the samples at different developmental stages were freshly frozen in a Sample Protector (Takara, Tokyo, Japan) at −80°C until RNA isolation.
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T. pui samples were collected in the Tibetan Plateau Peculiar Bio-resources Research Station of Sun Yat-sen University at Nyingchi in the Tibet Autonomous Region (4,156 m altitude, 29°36′N, 94°36′E) from June to August in 2014. The larvae were reared as previously described (Sun et al. 2011 (link), Wu et al. 2015 (link)). The larvae of different instars (third through eighth), female pupae of different ages (1, 9, 17, and 33 d old), and 1-d-old adult females were used for the analysis of gene expression. All of the samples at different developmental stages were freshly frozen in a Sample Protector (Takara, Tokyo, Japan) at −80°C until RNA isolation.
2
Metagenomic Analysis of Rat Gut Microbiome
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Fresh fecal contents were directly collected from the rat's cecum at the end of the study and stored in Sample Protector (TaKaRa, Dalian, China) at −80°C. The MoBio Power Fecal DNA Isolation kit (Mo BioLaboratories, Carlsbad, CA, USA) was used for DNA extraction. The quality of the extracted DNA was examined by agarose gel electrophoresis, and the OD 260/280 was analyzed by spectrophotometry. DNA libraries were prepared from 2 μg of total DNA for each sample using TruSeq DNA LT Sample Prep Kit v2 (Illumina, San Diego, California). Metagenomic sequencing was performed on HiSeq 3000 platform (Illumina, San Diego, California). After removing adapters, the raw reads were filtered to remove low-quality reads and reads that belong to the host. These high-quality reads from the samples were then assembled to contigs using Meta-Velevt. MetaGeneMark was employed to predict open reading frames (ORFs). In addition, a metagenomic catalog was generated based on the samples obtained in this study. Furthermore, the high-quality clean paired-end reads from each sample were aligned by BWA version 0.5.7-6 to the reference genes. Then the relative abundance of genes was predicted as described previously [15 (link)].
3
Immunization of Grass Carp: Sampling Protocol
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Five fishes were randomly sampled from each group at week 2, 4, and 6 after the beginning of the immunization. Grass carp was euthanized with overdose of eugenol mixture, and then skin mucus, blood, gallbladder, intestinal mucus, head kidney, and spleen were collected as described method (Guo et al. 2016 (link)). Briefly, skin mucus was gently scraped with a glass slide, then diluted with 0.5 ml sterile PBS, centrifuge at 4 °C, 4500 rpm for 20 min, and stored in −80 °C (Tang et al. 2017 (link)). Blood was collected from the caudal vein using a 1 ml injector, clotted in the room temperature for 2 h, and then sera were separated by centrifugation (4 °C, 4000 rpm for 15 min) and stored in − 20 °C until use (Jiang et al. 2017 (link)). The intestine was aseptically isolated, lavaged with 0.5 ml sterile PBS for several times, and centrifugated (4 °C, 4500 rpm for 20 min). The supernatant of the lavage fluid was stored at − 20 °C. The spleen and head kidney of each fish was dissected and preserved in sample protector (Takara Bio, Otsu, Japan) in − 80 °C. About 8 weeks after the beginning of administration, another three fishes in each group were sampled and euthanized, and the whole intestines of the three fishes were aseptically excised for gut microbiota analysis.
4
Coral Sampling and Molecular Analysis
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Coral samples were collected at Xiane reef, Nansha Islands, South China Sea, in June 2016 (Figure 1 ). Four coral species (unbleached coral and bleached coral collected from the same coral colony), Acropora tenuis (AC-H, AC-W), Goniastrea minuta (GM-H, GM-W), Pocillopora verrucosa (PV-H, PV-W) and Pocillopora meandrina (PM-H, PM-W) were sampled. The field sampling process of coral samples was showed in Figure 2 . Water temperatures in the sampling area ranged from 30.5 to 31°C. Three replicate samples of unbleached and bleached parts of coral (including tissue, mucus and skeleton) were collected using a hammer and chisel. Once collected, the coral samples (1 cm × 1 cm) were washed with sterile seawater three times to remove any surface attachments. Each sample was divided into two parts and placed in sterile centrifuge tubes with Sample Protector (Takara, Japan) for DNA and RNA extraction. DNA and RNA isolation of replicate samples was mixed and conducted using DNeasy and RNeasy plant mini kit (Qiagen, Germany) following the manufacturer’s instructions. Coral tissues were removed with an airbrush for the identification of each species. All coral samples were identified according to their ecological and morphological characteristics.
5
Transcriptomic Profiling of Przewalski's Lizard
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Six different tissue types (brain, liver, heart, muscle, and testicle/ootheca) from six individuals (three males and three females) of each species were sampled in order to obtain as many expressed genes as possible. Samples of P. przewalskii were collected from the vicinity of Yinchuan City, China (106.87°E, 38.32°N) with an altitude of 1,153 m a.s.l. Samples of P. vlangalii were collected from the Zoige County, Sichuan Province, China (102.48°E, 33.72°N) with an altitude of 3,464 m a.s.l. Lizards were captured by hand and euthanized on-site by intracoelomic injection of overdose pentobarbital solution, typically within one hour of capture. Tissue samples were removed and stored in Sample Protector (Takara) immediately following euthanasia and dissection.
6
Flounder HIRRV Challenge Protocol
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After the last sampling at 8th week, all fish were transferred to tanks equipped with a refrigerating apparatus and adapted to 14 °C by gradual decrease of water temperature. One week later, all the fish of four experimental groups were intraperitoneally injected with 100 µl of prepared virus suspension (106.8 TCID50/fish) according to the lethal dose (LD50) in flounder [50 (link)]. After challenge, thirty fish were randomly selected from each experimental group for monitoring mortality. Mortality was recorded daily for 14 d post-challenge and RPS was calculated using the following formula: (1−% mortality of vaccinated fish/% mortality of control fish) × 100. Another thirty fish were randomly selected from each experimental group for monitoring the HIRRV proliferation in the spleen of infected flounder. For sampling, three fish from each group were sacrificed on day 1 and day 3 post-injection, and the spleens were sampled and stored in Sample Protector (Takara) at − 80 °C until usage.
7
Transcriptome and SNP Analysis of Asiatic Toads
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For transcriptome sequencing, samples of Asiatic toads were collected from a low-altitude site (Chengdu, China, 104.01°E, 30.91°N, 559 m) and a high-altitude site (Zoige, China, 102.48°E, 33.72°N, 3464 m; Fig. 1 ). Eight individuals (four males and four females) were collected from each site by hand, and six different tissues (brain, liver, heart, muscle, and testicle/ootheca) were collected from each individual. Tissue samples were stored in Sample Protector (Takara) immediately following euthanasia and dissection.
Samples for SNP genotyping were collected from five sites along two altitudinal gradient transects, and 20 individuals were captured from each site. Three sites, Chengdu (104.01°E, 30.91°N, 559 m), Jiuzhaigou (104.15°E, 33.08°N, 1717 m), and Zoige (102.48°E, 33.72°N, 3464 m) are located in the Minshan mountain range and form the first transect. Two sites, Luding (102.24°E, 29.80°N, 1465 m) and Kangding (101.87°E, 30.27°N, 3072 m), are located in the Daxueshan mountain range and form the second transect. A toe from each individual was collected and preserved in 95 % ethanol. A map with all sampling sites is presented in Fig.1 .
Samples for SNP genotyping were collected from five sites along two altitudinal gradient transects, and 20 individuals were captured from each site. Three sites, Chengdu (104.01°E, 30.91°N, 559 m), Jiuzhaigou (104.15°E, 33.08°N, 1717 m), and Zoige (102.48°E, 33.72°N, 3464 m) are located in the Minshan mountain range and form the first transect. Two sites, Luding (102.24°E, 29.80°N, 1465 m) and Kangding (101.87°E, 30.27°N, 3072 m), are located in the Daxueshan mountain range and form the second transect. A toe from each individual was collected and preserved in 95 % ethanol. A map with all sampling sites is presented in Fig.
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