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Authors: Darach Miller
Proper citation: Darach Miller 2016. RNA extraction, small filters. protocols.io dx.doi.org/10.17504/protocols.io.feqbjdw Copy
Authors: Fatma Gomaa, Zuhong Li, Roberto Docampo, Peter Girguis, Virginia Edgcomb
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: B. saltans cells were electroporated using a square-wave electroporator (Nepa21, Bulldog Bio, Inc.) using one poring pulse of 200 volts with a pulse duration of 25 ms and five transfer pulses of 60 volts with a pulse duration of 99 ms, with plasmid targeting the 18S region (18S-GFP). A schematic representation of the plasmid, the target locus and the expected site of integration into the B. saltans genome is shown in Figure 1. Electroporated cells were selected with 1 µg/ml of G418, added 24 hours after electroporation. Cells were washed and subcultured into fresh selection medium every 3-4 days. G418 resistant cells started to emerge 7-9 days post-electroporation. Cells were processed for genotyping analysis to confirm plasmid integration 3 weeks post-electroporation. DNA was extracted from pools of transfected and wild cells using the Qiagen DNeasy Blood & Tissue kit. PCR analyses were used to characterize the 18S-GFP tagging using 6 sets of PCR primers, as shown in Figure 1 C. Gel electrophoresis image (Figure 2) showing the amplified PCR products at the expected sizes. Amplified PCR#1 with primer sets Ribo_tag_forward & GFP reverse (800 bp)Amplified PCR #2 with primer sets Neo_forward & Ribo_tag_reverse (1000 bp)Amplified PCR #3 with primer sets TubR & IG forward ( 3 bands)Amplified PCR #4 with primer sets Tub_forward & IG reverse (3 bands)Amplified PCR #5 with primer sets Ribo_tag_forward & Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)Electroporated cells were selected with 1 µg/ml of G418, added 24 hours after electroporation. Cells were washed and subcultured into fresh selection medium every 3-4 days. G418 resistant cells started to emerge 7-9 days post-electroporation. Cells were processed for genotyping analysis to confirm plasmid integration 3 weeks post-electroporation. DNA was extracted from pools of transfected and wild cells using the Qiagen DNeasy Blood & Tissue kit. PCR analyses were used to characterize the 18S-GFP tagging using 6 sets of PCR primers, as shown in Figure 1 C. Gel electrophoresis image (Figure 2) showing the amplified PCR products at the expected sizes. Amplified PCR#1 with primer sets Ribo_tag_forward & GFP reverse (800 bp)Amplified PCR #2 with primer sets Neo_forward & Ribo_tag_reverse (1000 bp)Amplified PCR #3 with primer sets TubR & IG forward ( 3 bands)Amplified PCR #4 with primer sets Tub_forward & IG reverse (3 bands)Amplified PCR #5 with primer sets Ribo_tag_forward & Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)Cells were processed for genotyping analysis to confirm plasmid integration 3 weeks post-electroporation. DNA was extracted from pools of transfected and wild cells using the Qiagen DNeasy Blood & Tissue kit. PCR analyses were used to characterize the 18S-GFP tagging using 6 sets of PCR primers, as shown in Figure 1 C. Gel electrophoresis image (Figure 2) showing the amplified PCR products at the expected sizes. Amplified PCR#1 with primer sets Ribo_tag_forward & GFP reverse (800 bp)Amplified PCR #2 with primer sets Neo_forward & Ribo_tag_reverse (1000 bp)Amplified PCR #3 with primer sets TubR & IG forward ( 3 bands)Amplified PCR #4 with primer sets Tub_forward & IG reverse (3 bands)Amplified PCR #5 with primer sets Ribo_tag_forward & Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)PCR analyses were used to characterize the 18S-GFP tagging using 6 sets of PCR primers, as shown in Figure 1 C. Gel electrophoresis image (Figure 2) showing the amplified PCR products at the expected sizes. Amplified PCR#1 with primer sets Ribo_tag_forward & GFP reverse (800 bp)Amplified PCR #2 with primer sets Neo_forward & Ribo_tag_reverse (1000 bp)Amplified PCR #3 with primer sets TubR & IG forward ( 3 bands)Amplified PCR #4 with primer sets Tub_forward & IG reverse (3 bands)Amplified PCR #5 with primer sets Ribo_tag_forward & Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)Gel electrophoresis image (Figure 2) showing the amplified PCR products at the expected sizes. Amplified PCR#1 with primer sets Ribo_tag_forward & GFP reverse (800 bp)Amplified PCR #2 with primer sets Neo_forward & Ribo_tag_reverse (1000 bp)Amplified PCR #3 with primer sets TubR & IG forward ( 3 bands)Amplified PCR #4 with primer sets Tub_forward & IG reverse (3 bands)Amplified PCR #5 with primer sets Ribo_tag_forward & Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)Amplified PCR#1 with primer sets Ribo_tag_forward & GFP reverse (800 bp)Amplified PCR #2 with primer sets Neo_forward & Ribo_tag_reverse (1000 bp)Amplified PCR #3 with primer sets TubR & IG forward ( 3 bands)Amplified PCR #4 with primer sets Tub_forward & IG reverse (3 bands)Amplified PCR #5 with primer sets Ribo_tag_forward & Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)Figure 1: Schematic representation of the (A); the 18S- GFP plasmid; (B) Ribosomal operon in B. saltans genome (C); and the expected site of plasmid integration in B. saltans genome through homologous regions 1 and 2 (HR1, HR2).Figure 2: Agarose gel electrophoresis image of the amplified PCR products for B. saltans cells transfected with 18S-GFP Cassette (S1 and S2) and the wild type cells (C). The primers sets and the expected product sizes are mentioned above in the text.Figure 3: B. saltans cells transfected with 18S-GFP tagging cassette. On the left: a light microscopy image showing the B. saltans cells (around 4 cells at different level). On the right: GFP fluorescent signal detected in the transfected cell. Scale bar 10 μm.
Proper citation: Fatma Gomaa, Zuhong Li, Roberto Docampo, Peter Girguis, Virginia Edgcomb 2018. Generating Stable Transfection in Bodo saltans. protocols.io dx.doi.org/10.17504/protocols.io.sh3eb8n Copy
Authors: Changwei Shao, Chang Li, Na Wang, Yating Qin, Wenteng Xu, Qun Liu, Qian Zhou, Yong Zhao, Xihong Li, Shanshan Liu, Xiaowu Chen, Shahid Mahboob, Xin Liu, Songlin Chen
Group: GigaScience Press, BGI
Summary: Background: The spotted sea bass (Lateolabrax maculatus) is a valuable commercial fish that is widely cultured in China. While analyses using molecular markers and population genetics have been conducted, genomic resources are lacking.Finding: Here, we report a chromosome-scale assembly of the spotted sea bass genome by high-depth genome sequencing, assembly and annotation. The genome scale was 0.62 Gb with contig and scaffold N50 length to be 31 Kb and 1,040 Kb, respectively. Hi-C scaffolding of the genome resulted in 24 pseudochromosomes containing 77.68% of the total assembled sequences. A total of 132.38Mb repeat sequences were detected, accounting for 20.73% of the assemble genome. 22,015 protein-coding genes were predicted, of which 96.52% were homologous to proteins in various databases. In addition, we constructed a phylogenetic tree using 1,586 single-copy gene families and identified 125 unique gene families in the spotted sea bass genome.Conclusions: We assembled a spotted sea bass genome, which will be a valuable genomic resource to understand the biology of the spotted sea bass, and will also lead to the development of molecular breeding techniques to generate spotted sea bass with better economical traits.
Proper citation: Changwei Shao, Chang Li, Na Wang, Yating Qin, Wenteng Xu, Qun Liu, Qian Zhou, Yong Zhao, Xihong Li, Shanshan Liu, Xiaowu Chen, Shahid Mahboob, Xin Liu, Songlin Chen 2018. Protocols for chromosome-level genome assembly of the spotted sea bass, Lateolabrax maculatus. protocols.io dx.doi.org/10.17504/protocols.io.ssxeefn Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The following is a "typical" restriction endonuclease reaction. Please see the "guidelines" tab below for the NEB tips on optimizing restriction digests.
Proper citation: New England Biolabs 2018. Restriction Digest. protocols.io dx.doi.org/10.17504/protocols.io.isycefw Copy
Authors: Nadia Szeinbaum, Yael Toporek
Proper citation: Nadia Szeinbaum, Yael Toporek 2018. Outreach: "Is This Alive" station (designed for and used at AbGradCon 2018). protocols.io dx.doi.org/10.17504/protocols.io.ra2d2ge Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the procedures for intracardiac perfusion fixation of postnatal mice, including anesthesia, exsanguination, fixation, brain removal and post-fixation storage. Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Proper citation: Allen Institute for Brain Science 2020. Mouse Cardiac Perfusion Fixation and Brain Collection. protocols.io dx.doi.org/10.17504/protocols.io.bd8vi9w6 Copy
Authors: Ms Alex Aitken
Group: VERVE Net, Sullivan Lab
Summary: This shows how to make 1M Tris.
Proper citation: Ms Alex Aitken 2016. 1M Tris. protocols.io dx.doi.org/10.17504/protocols.io.c5vy65 Copy
Authors: Marzia Ognibene
Proper citation: Marzia Ognibene 2020. Cell migration assay or Transwell assay. protocols.io dx.doi.org/10.17504/protocols.io.bgd9js96 Copy
Authors: Benito Recio Totoro
Summary: A collection of protocols we use routinely in the lab to produce and purify ookinetes of Plasmodium berghei.
Proper citation: Benito Recio Totoro 2020. Culture and purification of Plasmodium berghei ookinetes. protocols.io dx.doi.org/10.17504/protocols.io.bmpgk5jw Copy
Authors: Shevan Wilkin, Richard Hagan, Sandra Hebestreit, Madeleine Bleasdale, Ayushi Nayak, Li Tang, Traci N Billings, Nicole Boivin, Kristine Richter
Summary: Dental calculus from archaeoloigcal samples is now commonly extracted for palaeoproteomic analysis. This protocol combines an EDTA based pretreatment and a SP3 (single-pot, solid-phase-enhanced) protein extraction to prepare samples for LC-MS/MS analysis. This protocol is for moderate to well preserved archaeological calculus samples with a starting weight of 5-15 mg of calculus. We recommend batch sizes of 2-10 calculus samples, 1 extraction control (we use a powdered bone sample from an achaeological sheep bone as this should not be modern material), and 1 extraction blank. For the first time doing the protocol in a lab, we recommend using flanking blanks to identify any contaminants as from handling or from solutions.This protocol is based upon the original SP3 protocol published in Hughes et al. (2014) and the developments for palaeoproteomics of bone (Cleland et al., 2018). When using please cite the following three publications the protocol is based upon as well as the DOI for this protocol.Hughes, C. S., Foehr, S., Garfield, D. A., Furlong, E. E., Steinmetz, L. M., & Krijgsveld, J. (2014). Ultrasensitive proteome analysis using paramagnetic bead technology. Molecular Systems Biology, 10, 757. https://doi.org/10.15252/msb.20145625Hughes, C. S., Moggridge, S., Müller, T., Sorensen, P. H., Morin, G. B., & Krijgsveld, J. (2019). Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nature Protocols, 14(1), 68–85. https://doi.org/10.1038/s41596-018-0082-xCleland, T. P. (2018). Human Bone Paleoproteomics Utilizing the Single-Pot, Solid-Phase-Enhanced Sample Preparation Method to Maximize Detected Proteins and Reduce Humics. Journal of Proteome Research, 17(11), 3976–3983. https://doi.org/10.1021/acs.jproteome.8b00637
Proper citation: Shevan Wilkin, Richard Hagan, Sandra Hebestreit, Madeleine Bleasdale, Ayushi Nayak, Li Tang, Traci N Billings, Nicole Boivin, Kristine Richter 2020. SP3 (Single-Pot, Solid-Phase, Sample-Preperation) Protein Extraction for Dental Calculus. protocols.io dx.doi.org/10.17504/protocols.io.bfgrjjv6 Copy
Authors: Hebert F.O., Grambauer S., Barber I., Landry C.R., Aubin-Horth N.
Group: GigaScience Press
Summary: This protocol describes how to extract total RNA from flatworms. It is from:Hebert, F, O; Grambauer, S; Barber, I; Landry, C, R; Aubin-Horth, N (2016): Reference transcriptome sequence resource for the study of the Cestode Schistocephalus solidus, a threespine stickleback parasite. GigaScience Database. http://dx.doi.org/10.5524/100197
Proper citation: Hebert F.O., Grambauer S., Barber I., Landry C.R., Aubin-Horth N. 2016. RNA extraction protocol (Trizol). protocols.io dx.doi.org/10.17504/protocols.io.ew7bfhn Copy
Authors: avinash.kale
Group: UM-DAE Centre for Excellence in Basic Sciences
Proper citation: avinash.kale 2020. De-salting of tryptic digested peptides. protocols.io dx.doi.org/10.17504/protocols.io.bjdqki5w Copy
Authors: Juan Wang
Summary: Background: Aberrant DNA methylation occurs frequently in cancer. The aim of this study was to identify novel methylation markers in lung cancer in Xuanwei, China, through integrated genome-wide DNA methylation and gene expression studies.Methods: Differentially methylated regions (DMRs) and differentially expressed genes (DEGs) were detected on 10 paired lung cancer tissues and noncancerous lung tissues by methylated DNA immunoprecipitation combined with microarray (MeDIP-chip) and gene expression microarray analyses, respectively. Integrated analysis of DMRs and DEGs was performed to screen out candidate methylation-related genes. Both methylation and expression changes of the candidate genes were further validated and analyzed.Results: Compared with normal lung tissues, lung cancer tissues expressed a total of 6,899 DMRs, including 5,788 hypermethylated regions and 1,111 hypomethylated regions. Integrated analysis of DMRs and DEGs identified 45 tumor-specific candidate genes: 38 genes whose DMRs were hypermethylated and expression was downregulated, and 7 genes whose DMRs were hypomethylated and expression was upregulated. The methylation and expression validation results identified 4 candidate genes (STXBP6, BCL6B, FZD10, and HSPB6) that were significantly hypermethylated and downregulated in most of the tumor tissues compared with the noncancerous lung tissues.Conclusions: This integrated analysis of genome-wide DNA methylation and gene expression in lung cancer in Xuanwei revealed several genes regulated by promoter methylation that have not been described in lung cancer before. These results provide new insight into the carcinogenesis of lung cancer in Xuanwei and represent promising new diagnostic and therapeutic targets.
Proper citation: Juan Wang 2018. Integrated analysis of DNA methylation profiling and gene expression profiling identifies novel markers in lung cancer in Xuanwei, China. protocols.io dx.doi.org/10.17504/protocols.io.rsxd6fn Copy
Authors: Rene Flores Clavo, Cristian Daniel Asmat Ortega, Nataly Ruiz Quinones
Group: Metabolomics Protocols & Workflows, RENE FLORES
Proper citation: Rene Flores Clavo, Cristian Daniel Asmat Ortega, Nataly Ruiz Quinones 2021. Mass Spectrometry analysis and Molecular MS/MS network. protocols.io dx.doi.org/10.17504/protocols.io.br2fm8bn Copy
Authors: Kendra Negrey, Meredith Howard, Jayme Smith, Raphael Kudela, David Caron
Summary: SPATT has been developed and tested for a variety of resins (see review by Kudela, 2017). These Standard Operating Procedures (SOP) outline the use of the resin DIAON HP20 since it has been demonstrated to quantify microcystins, anatoxin-a, saxitoxin, domoic acid, and okadaic acid in fresh, brackish, and marine waters, all from the same SPATT sampling device (Lane et al. 2010; Miller et al. 2010, Kudela 2011, Gibble and Kudela, 2014, Howard et al., 2017, Kudela, 2017, Peacock et al., 2018). Cylindrospermopsin and nodularin have also been detected using SPATT with DIAON HP20 resin in California, however, the use of DIAON HP20 for these toxins has not been well characterized in the laboratory. This methodology is ready for wider adoption by the research, monitoring, and management communities interested in detecting and tracking the dynamics of freshwater and marine toxins. The SOP described herein for the assemblage and construction of SPATT bags can be used for deployment in freshwater, brackish and marine environments and the extraction process described below is for the analysis of both marine toxins and cyanotoxins.
Proper citation: Kendra Negrey, Meredith Howard, Jayme Smith, Raphael Kudela, David Caron 2019. Standard Operating Procedure for Solid Phase Adsorption Toxin Testing (SPATT) Assemblage and Extraction of HAB Toxins. protocols.io dx.doi.org/10.17504/protocols.io.xkpfkvn Copy
Authors: Emma Betteridge, Naomi Park, Keith James, Jillian Durham, Josh Quick
Group: Coronavirus Method Development Community
Summary: This SOP describes the procedure for generating cDNA from SARS-CoV-2 viral nucleic acid extracts and subsequently producing 275nt amplicons. This is followed by library construction, equivolume pooling of samples and quantitation, prior to sequencing on the Illumina NovaSeq. A key benefit of the short amplicon method is that it can be utilised on any Illumina platform with a 300 cycle (or greater) kit.It is an adaptation of the 400nt COVID-19 ARTIC v3 amplicon protocol which can be found here:https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bgxjjxknBoth the above protocols were adapted from the nCov-2019 sequencing protocol: https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w
Proper citation: Emma Betteridge, Naomi Park, Keith James, Jillian Durham, Josh Quick 2020. COVID-19 ARTIC v3 Illumina library construction and sequencing protocol - short amplicons (275bp). protocols.io dx.doi.org/10.17504/protocols.io.bh4zj8x6 Copy
Authors: Brian Smith, [email protected] Micro.tucson
Summary: This protocol will make one liter of salt water LB. Adjust volumes as necessary.
Proper citation: Brian Smith, [email protected] Micro.tucson 2019. SW-LB Media (1L). protocols.io dx.doi.org/10.17504/protocols.io.xf2fjqe Copy
Authors: Ratha-Korn Vilaichone, Natsuda Aumpan
Proper citation: Ratha-Korn Vilaichone, Natsuda Aumpan 2020. p53 immunohistochemistry protocol. protocols.io dx.doi.org/10.17504/protocols.io.bigjkbun Copy
Authors: Anand Patel, Asa Karlstrom, Brittney Gordon, Elizabeth Stewart, Michael Dyer
Group: NCIHTAN
Summary: This protocol was used for the dissociation of fresh pediatric neuroblastoma samples from patient tumors or orthotopic patient derived xenografts. Using this method, we were able to prepare highly viable (>90%) single-cell suspensions compatible with droplet-based single-cell RNA-Seq technology (Slyper et al).For the Human Tumor Atlas Pilot Project (HTAPP), this protocol has been successfully applied to neuroblastoma resections collected from abdominal and paraspinal sites.Description of this protocol and guidance for testing and selecting methods for processing other tumor and sample types can be found in Slyper et al.
Proper citation: Anand Patel, Asa Karlstrom, Brittney Gordon, Elizabeth Stewart, Michael Dyer 2020. HTAPP_Dissociation of human neuroblastoma tumors to a single-cell suspension for single-cell RNA-seq using Papain. protocols.io dx.doi.org/10.17504/protocols.io.98ah9se Copy
Authors: Sricharan Kadimi
Group: UConn iGEM
Summary: A protocol kindly provided to our team by the advisor to the 2018 MIT iGEM team (Dr. Brian Teague). It is modified slightly from that version (made by Stefan Maas in 1997) to fit our needs. Stefan Maas' protocol is similar to the Hanahan, D. protocol from 1983.It worked pretty well to make good competent cells in Summer 2018 and we just used it again in Summer 2019.
Proper citation: Sricharan Kadimi 2019. RbCl Uber-Competent Cells. protocols.io dx.doi.org/10.17504/protocols.io.3hygj7w Copy
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