Protocols   Select Another Resource Report Type

Authors: Helena Pound, Steven Wilhelm
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), Great Lakes Center for Fresh Waters and Human Health
Summary: Find genes of interest in assembled metatranscriptomic library using blast.

Proper citation: Helena Pound, Steven Wilhelm 2019. Metatranscriptomic screening for genes of interest. protocols.io https://dx.doi.org/10.17504/protocols.io.7vyhn7w Copy   

•   Source:

Authors: Rosey Bayne, Stefan Bresson, David Tollervey, Edward Wallace
Group: Wallace lab for Fungal RNA
Summary: CRAC (Cross-linking and cDNA Analysis) is a method that uses UV-crosslinking to identify which RNAs bind to an RNA-Binding Protein of interest and the specific sites in the RNA where this binding occurs. CRAC is one of the family of UV crosslinking and immunoprecipitation (CLIP) protocols, reviewed by Lee and Ule (DOI: 0.1016/j.molcel.2018.01.005).The protein is tagged with with a tandem affinity tag: in this protocol we use an HTP tag (His6-TEV-ProteinA) that allows immuno-affinity purification of the protein with its cross-linked RNAs by binding of the ProteinA moiety of the tag to IgG Sepharose columns. After washing, the protein is then released from the column by cleavage with TEV protease and subjected to nickel-affinity purification through the His6 moiety under highly denaturing conditions. This allows accurate mapping of RNA binding sites (Bohnsack, et al, DOI: 10.1016/B978-0-12-396546-2.00013-9) ; Granneman, S. et al (DOI: 10.1073/pnas.0901997106 ). The protocol can also be adapted for use with HTF (His6-TEV-FLAG3) tags (Tree, J.J. et al,  DOI: 10.1016/j.molcel.2014.05.006) or HF (His8-Ala4-FLAG) tags (Bresson, S., Shchepachev, V., Spanos, C., Turowski, T., Rappsilber, J. and Tollervey, D.(2020): DOI: /10.1101/2020.05.14.096354) by replacing the IgG sepharose column with anti-FLAG magnetic beads and elution with excess FLAG peptide, removing the need for TEV cleavage which can be inefficient. The cDNA libraries generated allow a transcriptome -wide analysis of the interactome of the protein.

Proper citation: Rosey Bayne, Stefan Bresson, David Tollervey, Edward Wallace 2020. CRAC Analysis in Budding Yeast with HTP Tagged Proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.5ppg5mn Copy   

•   Source:

Authors: Josh Quick
Group: ARTIC

Proper citation: Josh Quick 2019. Ebola virus sequencing protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7nwhmfe Copy   

•   Source:

Authors: Madeline M Glennon, Austin Skinner, Mara Krutsinger, Marino Resendiz

Proper citation: Madeline M Glennon, Austin Skinner, Mara Krutsinger, Marino Resendiz 2020. Reverse transcription of complementary DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.birskd6e Copy   

•   Source:

Authors: Ji Won Choi, Duk Kyung Kim, Jin Kyoung Kim, Eun Jee Lee, Jea Youn Kim
Summary: Original dataset of the study entitled "A retrospective analysis on the relationship between intraoperative hypothermia and postoperative ileus after laparoscopic colorectal surgery"

Proper citation: Ji Won Choi, Duk Kyung Kim, Jin Kyoung Kim, Eun Jee Lee, Jea Youn Kim 2017. Original dataset. protocols.io https://dx.doi.org/10.17504/protocols.io.mbyc2pw Copy   

•   Source:

Authors: April Woods, G Jason Smith
Summary: Cell preservation and counting techniques for Monterey Wharf II monitoring and CA-HABMAP reporting.

Proper citation: April Woods, G Jason Smith 2018. Monterey Wharf II Cell Enumeration For HABMAP Reporting. protocols.io https://dx.doi.org/10.17504/protocols.io.pcbdisn Copy   

•   Source:

Authors: Heather Eckart, Ran Zhou
Group: Helmsley project_Basu lab
Summary: This protocol provides a procedure for human intestinal organoid dissociation into a single cell suspension and nuclei isolation prior to Single Cell ATAC-Sequencing.

Proper citation: Heather Eckart, Ran Zhou 2020. Intestinal Organoid Dissociation and Nuclei Isolation for Single Cell ATAC-Seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bdeui3ew Copy   

•   Source:

Authors: Eftychis Frangedakis, Susana Sauret-Gueto
Group: OpenPlant Project

Proper citation: Eftychis Frangedakis, Susana Sauret-Gueto 2019. Cloning of L0 parts into pUAP-ye for Loop typeIIS. protocols.io https://dx.doi.org/10.17504/protocols.io.4wdgxa6 Copy   

•   Source:

Authors: Sina Booeshaghi, Yeokyoung Kil, Kyung Hoi Min, Jase Gehring, Lior Pachter
Group: Pachter Lab
Summary: We present colosseum, a low-cost, modular, and automated fluid sampling device for scalable fluidic applications. The colosseum fraction collector uses a single motor, can be built for less than $100 using off-the-shelf and 3D-printed components, and can be assembled in less than an hour. Build Instructions and source files are available at https://github.com/pachterlab/colosseum.

Proper citation: Sina Booeshaghi, Yeokyoung Kil, Kyung Hoi Min, Jase Gehring, Lior Pachter 2021. Assembly Instructions for colosseum. protocols.io https://dx.doi.org/10.17504/protocols.io.btz3np8n Copy   

•   Source:

Authors: Kenneth Schackart, Kattika Kaarj
Group: 481b Laboratory
Summary: This protocol explains how to refresh the media of cultured mammalian cells grown in a tissue culture flask.

Proper citation: Kenneth Schackart, Kattika Kaarj 2019. Mammalian Cell Culture: Refreshing Media. protocols.io https://dx.doi.org/10.17504/protocols.io.xqifmue Copy   

•   Source:

Authors: New England Biolabs
Group: New England Biolabs (NEB)

Proper citation: New England Biolabs 2020. Protocol for T7 Exonuclease (NEB #M0263). protocols.io https:// Copy   

•   Source:

Authors: Chuandan Wan
Summary: A systematic literature search was performed using PubMed, Embase, and WANFANG MED ONLINE databases up to March 18, 2019. All literature was analyzed using Meta Disc 1.4 and STATA 14.0 software. Diagnostic measures of accuracy of ctDNA in cervical cancer were pooled and investigated. Fifteenstudies comprising 1109 patients with cervical cancer met our inclusion criteria and were subjected to analysis.

Proper citation: Chuandan Wan 2019. meta analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.8t4hwqw Copy   

•   Source:

Authors: Eric Deflesselle
Summary: Genomic DNA was extracted from circulating blood leucocytes by using Gentra Puregene Blood Kits according to the manufacturer’s protocol (Qiagen) and was stored at -20°C. The TRKB polymorphismswere genotyped by the IntegraGen company (Evry, France) using the Fluidigm®BioMarkTMHD system (Fluidigm Corporation, CA, USA), using TaqMan allelic discrimination (Coulbault et al., 2006), with the ABI Prism® 7900HT Sequence Detection Sys­tem (Life Technologies).

Proper citation: Eric Deflesselle 2018. METADAP genetic protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.ti2ekge Copy   

•   Source:

Authors: Angel Justiz-Vaillant
Summary: Chemical synthesis facilitates the generation of peptides which are exceedingly difficult to express in bacteria, peptide/protein backbone modification, the incorporation of unnatural amino acids, and the production or synthesis of D-proteins. The C-terminal cysteine can be added to the amino acidic sequences of HIV peptides (fragment 579-601 of the HIV-gp41 [1]. These peptide fragments were dimerized by cysteine oxidation with dimethyl-sulfoxide [2] to facilitate their conjugation to keyhole limpet hemocyanin that acts as a carrier protein. Reference:McPhee DA, Kemp BE, Cumming S, Stapleton D, Gust ID, Doherty RR. Recognition of envelope and tat protein synthetic peptide analogs by HIV positive sera or plasma. FEBS Lett. 1988;233(2):393-396. doi:10.1016/0014-5793(88)80468-x. Tam JP, Wu CR, Liu w, Zhang JW (1991) Disulfide bond formation in peptides by dimethyl sulfoxide. Scope and applications. J Am Chem Soc 113: 66576662.

Proper citation: Angel Justiz-Vaillant 2020. Peptide fragment (579-601 from HIV-gp41) conjugated to keyhole limpet haemocyanin to be used as HIV immunogen.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjibkkan Copy   

•   Source:

Authors: Germán Alberto Téllez Ramírez, Lily Johanna Toro, Juliana Franco Castrillon, Diana Carolina Henao, Jhon Carlos Castaño Osorio, Grupo De Inmunología Molecular Universidad Del Quindio
Summary: Este método es basado en el método clásico de micro-dilución en placa de NCLSS (National Committee of Laboratory Safety and Standards (NCLSS) como fue publicado en Amsterdam, D. 1996. Susceptibility testing of Antimicrobials in liquid media. In 'Antibiotics in Laboratory Medicine', Lorian, V., ed. Fourth Edition, pp.52-111. Williams and Wilkins, Baltimore.  Modificado con respecto a la lectura de la placa utilizando un indicador metabólico para incrementar la sensibilidad de la prueba respecto al crecimiento de las bacterias.La realizacion de este protocolo fue posible gracias al apoyo del departamento administrativo de ciencia tecnología e innovacion, Colciencias a traves del proyecto 111356933173 convocatoria569-2012. Este protocolo fue estandarizado con el desarrollo del proyecto de grado "OPTIMIZACIÓN DE UN MÉTODO PARA EVALUAR LA ACTIVIDAD ANTIBACTERIANA IN VITRO EN MICROVOLÚMENES" de la estudiante Juliana Franco para optar por el titulo de Biologa.

Proper citation: Germán Alberto Téllez Ramírez, Lily Johanna Toro, Juliana Franco Castrillon, Diana Carolina Henao, Jhon Carlos Castaño Osorio, Grupo De Inmunología Molecular Universidad Del Quindio 2020. MICRO-DILUCIÓN EN PLACA PARA LA EVALUACIÓN DE LA ACTIVIDAD ANTIMICROBIANA EN 50 uL . protocols.io https://dx.doi.org/10.17504/protocols.io.8xmhxk6 Copy   

•   Source:

Authors: Luiz Filipe Bartoleti, Luiz Filipe Bartoleti
Summary: Protocol used to extract whole genome DNA from Nephila clavipes (Araneae: Araneidae) specimens.

Proper citation: Luiz Filipe Bartoleti, Luiz Filipe Bartoleti 2017. DNA extraction of Nephila clavipes using the Wizard Genomic DNA Purification kit (Promega) . protocols.io https://dx.doi.org/10.17504/protocols.io.kbhcsj6 Copy   

•   Source:

Authors: Rob A B Oostendorp

Proper citation: Rob A B Oostendorp 2018. 1. Flowchart illustrating the steps of the clinical reasoning process. protocols.io https://dx.doi.org/10.17504/protocols.io.uscewaw Copy   

•   Source:

Authors: Luciano Martelotto
Group: Human Cell Atlas Method Development Community, Single Cell Core, Harvard Medical School
Summary: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, and is an extension of the Frankenstein (D.O.F means Daughter Of Frankenstein). Developed to prepare nuclei isolates from fresh and frozen material of small-to-large sizes. The good thing is that it does not uses FACS but OptiPrep® continuous gradient to remove debris. It is the alternative protocol when FACS is not available. This protocol works better with smaller samples. After centrifugation the nuclei will form a pellet and contaminating membranes will float at the top. This continuous gradient can be applied for as many times as needed to the same prep to continue cleaning up your nuclei suspensions.

Proper citation: Luciano Martelotto 2021. Daughter of Frankenstein protocol for nuclei isolation from fresh and frozen tissues using OptiPrep® continuous gradient. protocols.io https://dx.doi.org/10.17504/protocols.io.bs99nh96 Copy   

•   Source:

Authors: Diep Ganguly
Group: Pogson Genomics Group
Summary: This protocol (based on the BigDye® Terminator v3.1 Cycle Sequencing Kit) is for performing terminator cycling sequencing reactions for Sanger sequencing of amplified PCR products or plasmid DNA on the 3130X genetic analyser (Applied Biosystems).

Proper citation: Diep Ganguly 2020. Dye-terminator DNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bhxmj7k6 Copy   

•   Source:

Authors: Yabing Cao
Summary: EGFR tyrosine kinase inhibitor (TKI) can significantly improve objective response rate (ORR) and prolong progression free survival (PFS) in EGFR mutated locally advanced or metastatic NSCLC patients compared to platinum-based chemotherapy, with less adverse events and higher quality of life (QoL).Osimertinib is an oral, potent, irreversible third-generation EGFR TKI that is selective for EGFR TKI sensitizing mutation and the T790M resistance mutation. Two phase 2 studies(AURA extension, AURA2)have demonstrated encouraging efficacy and tolerable toxicity.However, local data reflecting testing status including testing platform, sample tested, and turnaround time in China is still lacking. The current study will not only assess the effectiveness of osimertinib treatment in a real world setting, but will also help us to understand the real-world testing patterns among T790M mutation positive locally advanced or metastatic NSCLC patients who have progressed after EGFR TKI treatment.Objectives and Hypotheses:The objective of this study is to assess osimertinib effectiveness among locally advanced or metastatic T790M mutation positive NSCLC patients who have progressed after EGFR TKI therapy.The specific study objectives that will be assessed include the following:Primary Objective§ To assess the real world effectiveness of osimertinib treatment in locally advanced or metastatic NSCLC patients with a EGFR T790M mutation in terms of response rate (RR)Secondary Objectives§ To assess the real world effectiveness of osimertinib treatment in locally advanced or metastatic NSCLC patients with a EGFR T790M mutation in terms of progression free survival (PFS)§ To describe the pattern of T790M mutation testing, including turnaround time, sample type, testing techniques, EGFR subtype, T790M mutation abundance if tested quantitatively.§ To assess the real world safety profile of osimertinib§ To describe the treatment pattern including treatment lines and combination therapy (radiotherapy, surgery, etc.) in a real world setting

Proper citation: Yabing Cao 2019. Observational, Real World Study of Osimertinib for Patients with Locally advanced/Metastatic T790M Mutation-Positive An observational, retrospective study conducted among locally advanced or metastatic T790M mutation-positive NSCLC patients progressed afte. protocols.io https://dx.doi.org/10.17504/protocols.io.2qygdxw Copy   

•   Source: