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Authors: Francois-Yves bouget
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: This protocol describes the selection and growth of stable transformants in semi solid agarose medium. Developped initially for Ostreococcus tauri, it also works for Bathycoccus.
Proper citation: Francois-Yves bouget 2017. Selection of stable transformants in Ostreococcus tauri and Bathycoccus prasinos. protocols.io dx.doi.org/10.17504/protocols.io.hc4b2yw Copy
Authors: Tomasz Suchan
Group: Molecular Biogeography Group
Summary: Protocol for preparing SPRI beads 100x cheaper than the commercial AMPure XP.Based on DeAngelis et al. (1995) and online protocols:https://s3-us-west-2.amazonaws.com/oww-files-public/1/17/Serapure_bead_recipe.pdfhttps://s3-us-west-2.amazonaws.com/oww-files-public/f/f8/SPRI_buffers_v2_2.pdf
Proper citation: Tomasz Suchan 2019. SPRI purification beads. protocols.io dx.doi.org/10.17504/protocols.io.q3qdymw Copy
Authors: Samah El Ghamrasni
Group: Human Cell Atlas Method Development Community, CZI START Project
Summary: A protocol designed to dissociate fresh breast tissues (surgical specimens and biopsies) for single-cell RNAseq.
The protocol has been demonstrated to work successfully with fresh and cryopreserved tissues.
Proper citation: Samah El Ghamrasni 2019. Breast tumours dissociation . protocols.io dx.doi.org/10.17504/protocols.io.7m9hk96 Copy
Authors: Camila Rangel Smith
Group: Vivarium Population Spenser
Summary: Description of the steps followed by Vivarium Population Spenser library when running the Immigration module.
Proper citation: Camila Rangel Smith 2020. Vivarium Population Spender: Immigration Module. protocols.io dx.doi.org/10.17504/protocols.io.bn9dmh26 Copy
Authors: Payam Amiri
Proper citation: Payam Amiri 2017. Liquid Growth Media for Bacteria. protocols.io dx.doi.org/10.17504/protocols.io.hn8b5hw Copy
Authors: George Moberly
Summary: Prototype Protocol
Proper citation: George Moberly 2019. Prototype Protocol. protocols.io dx.doi.org/10.17504/protocols.io.73yhqpw Copy
Authors: Ruth Timme, Maria Balkey, William Wolfgang, Errol Strain
Group: GenomeTrakr
Summary: PURPOSE: Guidance on how to populate NCBI's Pathogen metadata package, maximizing interoporability for foodborne pathogen surveillance. SCOPE: This protocol provides detailed instructions for populating the core metadata fields within the Pathogen metadata package.
Proper citation: Ruth Timme, Maria Balkey, William Wolfgang, Errol Strain 2020. Populating the NCBI pathogen metadata template. protocols.io dx.doi.org/10.17504/protocols.io.bdwti7en Copy
Authors: Gage Moreno, David O'connor
Summary: This protocol outlines the methods to perform unbiased direct metagenomic sequencing of nucleic acid extracts from cell-free fluids. This protocol can be adapted to be run on Illumina and Nanopore sequencing platforms. The protocol is based off of the work from Kafetzopoulou et al. (PMID: 30563591). Liana has provided the lab with detailed protocols, and has worked with us extensively on optimizing and getting protocols running efficiently. Please note that this protocol has been updated to use SuperScript IV with it’s optimal temperature which has been reflected in Lewandowski et al. (DOI: https://doi.org/10.1128/JCM.00963-19)Notes:* This protocol has been used to sequence influenza direction from respiratory clinical samples (DOI: https://doi.org/10.1128/JCM.00963-19).* A team from China published last week on a 2019-nCoV familial cluster using the SISPA protocol as for coronavirus whole genome sequencing (DOI: https://doi.org/10.1016/S0140-6736(20)30154-9)SISPA-Primer A - 5'-GTT TCC CAC TGG AGG ATA-(N9)-3'SISPA-Primer B - 5′-GTT TCC CAC TGG AGG ATA-3′
Proper citation: Gage Moreno, David O'connor 2020. Sequence-Independent, Single-Primer Amplification of RNA viruses. protocols.io dx.doi.org/10.17504/protocols.io.bckxiuxn Copy
Authors: Elisa Gonzalez-Rothi, Latoya Allen, Alec Simon, Yasin Seven, Marissa Ciesla, Gordon Mitchell
Group: SPARC
Summary: This protocol describes the procedures for conducting long-term exposure to various intermittent hypoxia (or normoxia) protocols in rats.
Proper citation: Elisa Gonzalez-Rothi, Latoya Allen, Alec Simon, Yasin Seven, Marissa Ciesla, Gordon Mitchell 2019. SPARC Long-term exposure to intermittent hypoxia (or normoxia) using a custom in-cage computer controlled system. protocols.io dx.doi.org/10.17504/protocols.io.2j3gcqn Copy
Authors: Juho Eineluoto, Petrus Järvinen, Anu Kenttämies, Tuomas Kilpeläinen, Hanna Vasarainen, Kevin Sandeman, Andrew Erickson, Tuomas Mirtti, Antti Rannikko
Summary: Introduction: This study was conducted to describe the repeat multiparametric MRI (mpMRI)
changes occurring in prostate cancer (PCa) patients during active surveillance (AS), and to study
possible associations between mpMRI-related parameters in predicting prostate biopsy (Bx)
Gleason score (GS) upgrading >3+3 (GU) and protocol-based treatment change (TC).
Materials and methods: The study cohort consisted of 76 AS patients with GS 3+3 PCa
and at least two consecutive mpMRIs of the prostate performed between 2006 and 2015. Patients
were followed according to the Prostate Cancer Research International Active Surveillance (PRIAS) protocol and additional mpMRIs. The primary end points were GS upgrading (GU) (>3+3) in protocol-based biopsies and protocol-based TC.
Results: 53/76 (69%) patients had progression (PIRADS upgrade, size increase or new
lesion[s]), 18/76 (24%) had radiologically stable disease, and 5/76 (7%) had regression
(PIRADS or size decrease, disappearance of lesion[s]) in repeat mpMRIs during AS. PIRADS
scores 4 to 5 in the initial mpMRI was associated with GU (p=0.008) and protocol-based TC
(p=0.009). Tumour progression on repeat mpMRIs was associated with TC (p=0.045) but not with
GU (p=1.00). PIRADS scores 4-5 predict GU (sensitivity 0.80 [95% confidence interval (CI);
0.51-0.95, specificity 0.62 [95% CI; 0.52-0.77]) with PPV and NPV values of 0.34 (95% CI;
0.21-0.55) and 0.93 (95% CI; 0.80-0.98), respectively.
Conclusion: mpMRI is a useful tool not only to select but also to monitor PCa
patients on AS.
Proper citation: Juho Eineluoto, Petrus Järvinen, Anu Kenttämies, Tuomas Kilpeläinen, Hanna Vasarainen, Kevin Sandeman, Andrew Erickson, Tuomas Mirtti, Antti Rannikko 2017. Repeat multiparametric MRI in prostate cancer patients on active surveillance. protocols.io dx.doi.org/10.17504/protocols.io.j5jcq4n Copy
Authors: Timothy Gilpatrick, Isac Lee, James E. Graham, Etienne Raimondeau, Rebecca Bowen, Andrew Heron, Fritz J. Sedlazeck, Winston Timp
Summary: This protocol is designed to help users achieve targeted enrichment for regions of interest using nanopore sequencing, providing higher coverage for myriad future analysis applications. GuideRNA components (crRNA and tracrRNA) were designed using online tools provided by IDT.
Proper citation: Timothy Gilpatrick, Isac Lee, James E. Graham, Etienne Raimondeau, Rebecca Bowen, Andrew Heron, Fritz J. Sedlazeck, Winston Timp 2019. Cas9 Enrichment for Nanopore Sequencing. protocols.io dx.doi.org/10.17504/protocols.io.3acgiaw Copy
Authors: John Tompkins, Mike Andresen
Proper citation: John Tompkins, Mike Andresen 2019. Mouse Stellate- Intracellular recording. protocols.io dx.doi.org/10.17504/protocols.io.2qfgdtn Copy
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, hence the name Frankenstein. Developed to prepare nuclei isolates from small sample sizes (as little as a grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any debris, especially ambient RNA, to help reduce background. The reference protocols can be found in the following papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al.This protocol has been validated in the Single-Cell Innovation Lab (UMCCR) and other labs worldwide for single nuclei experiments using 10x Genomics technologies.The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, and cell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer, lymphoma, xenografts and other samples types.Cardiomyocytes can be really difficult to prepare but we have successfully prepare this. Get in touch for some tips.
Proper citation: Luciano Martelotto 2020. 'Frankenstein’ protocol for nuclei isolation from FRESH and FROZEN tissue for snRNA-Seq (10x Genomics Platform). protocols.io dx.doi.org/10.17504/protocols.io.bq27myhn Copy
Authors: Christian Voldby, Kent Green, Susanne Rosthøj, Thomas Kongstad, Lue Philipsen, Frederik Buchvald, Marianne Skov, Tania Pressler, Per Gustafsson, Kim G. Nielsen
Summary: It is presumed that early detection and treatment of the chronic lung diseases cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) are crucial for improving the prognosis. Mucus stagnation and secondary infections and inflammation in the airways causes progressive lung damage from infancy in CF and PCD, which again causes early death in CF and reduced pulmonary function in PCD. Early detection of ongoing lung damage will facilitate early intervention with intensive treatment, enhance disease surveillance and further improve preventive treatment. This will improve quality of life for patients with both CF and PCD and likely improve survival in CF.Pulmonary function is an important marker of prognosis and effect of treatment, but current pulmonary function tests do not register the earliest damages in the lungs in school children – and are inappliccable for infants and toddlers due to requirements for good coorporation. For this reason there is a demand for more sensitive methods that can be used in all age groups. Gas washout – multiple breath inert gas washout (MBW) – is a method that meets these criteria and seems promising for detection of minimal lung damage prior to debut of clinical symptoms and detection by other methods.MBW is for the first time in Denmark established in the Pediatric Pulmonary Service at Copenhagen University Hospital, where a recently established ”Respiratory Physiology Lab for Children” offers a wide range of methods to detect lung disease in children.Aim:The aim of the Ph.D.-project is to investigate:Reliability of results conducted from MBW measurements under different standardized conditions (reproducibility), including establishment of a Danish reference material for children.Whether MBW can be used as a marker of early lung disease compared to conventional lung function methods in the chronic lung diseases CF and PCD.Whether MBW can be used as a relevant outcome to detect interventions in basic CF treatment.The Ph.D.-project will generate a new and more aggressive approach to surveillance and treatment of patients with CF and PCD with an overall aim to reduce morbidity and mortality and improve quality of life. Furthermore, it is expected that this project will generate new knowledge that can be used in the more common lung diseases asthma and COPD, which affects 10% and 15% of children and adults, respectively.
Proper citation: Christian Voldby, Kent Green, Susanne Rosthøj, Thomas Kongstad, Lue Philipsen, Frederik Buchvald, Marianne Skov, Tania Pressler, Per Gustafsson, Kim G. Nielsen 2018. Influence of chest physiotherapy on mulitple breath washout in children with cystic fibrosis. protocols.io dx.doi.org/10.17504/protocols.io.jsjcncn Copy
Authors: Zhujun Wei
Group: 2020 iGEM NEFU China
Proper citation: Zhujun Wei 2020. Transformation. protocols.io dx.doi.org/10.17504/protocols.io.bnykmfuw Copy
Authors: Rona Ortenberg, Erez Nissim Baruch
Summary: This protocol allows to prepare paraffin block of any cell line or co-cultured cells and subsequently these blocks could be used for cell microarray construction. Slides are suitable for ICH/IF staining procedures.Cell microarray can be used, exactly like a tissue microarray, as a high-throughput tool for omics validation or investigation of functional dynamics of targets during a prolonged cell-cell interaction or any other treatments.
Proper citation: Rona Ortenberg, Erez Nissim Baruch 2019. Cell line block and microarray preparation. protocols.io dx.doi.org/10.17504/protocols.io.6pnhdme Copy
Authors: LI-COR Biosciences
Group: LI-COR Biosciences
Summary: QWB is often used for relative analysis of protein phosphorylation and other post- translational modifications (PTMs). This method combines two primary antibodies raised in different hosts: a phospho-specific or other PTM-specific antibody, and a pan-specific antibody that recognizes the target protein regardless of its modification state. Fluorescently-labeled secondary antibodies are used to simultaneously detect and discriminate the two signals. Phospho-specific signals are then normalized against the total level of the target protein, using that protein as its own internal loading control for maximum accuracy.
This multiplex fluorescent method is used to analyze changes in target protein phosphorylation, glycosylation, acetylation, ubiquitination, and other PTMs.
Note: This protocol is intended for use with near-infrared fluorescent Western blots.
Proper citation: LI-COR Biosciences 2017. Pan/Phospho Analysis For Western Blot Normalization. protocols.io dx.doi.org/10.17504/protocols.io.g8dbzs6 Copy
Authors: Alice Pawlowski
Summary: Standard protocol to prepare cells for highly efficient DNA uptake using CaCl2 (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 2001).
Proper citation: Alice Pawlowski 2020. Preparation of chemically competent E. coli cells. protocols.io dx.doi.org/10.17504/protocols.io.gtcbwiw Copy
Authors: Vivian Liu
Summary: This protocol described a easy procedure to purify anti-sera in small scale batch. Protein A or protein G beads can be used depending on the immunoglobulin types.
Proper citation: Vivian Liu 2015. Antibody Purification (small scale). protocols.io dx.doi.org/10.17504/protocols.io.dny5fv Copy
Authors: Nicola O'Reilly, Svend Kjaer, Maria Greco
Group: Coronavirus Method Development Community, Crick COVID-19 Consortium
Summary: Purpose of examination / Clinical relevanceAt the end of 2019, several pneumonia cases were reported in Wuhan, China and the pathogen was confirmed as a new viral strain. World Health organization has named the newly identified coronavirus as 2019-nCoV, also known as COVID19. The disease developed into a dangerous pandemic, posing major challenges to the NHS. Although more research is necessary to better understand the virus, in response to the emergency, simple and rapid testing is essential to identify the virus in infected individuals. This will aid the implementation of efficient interventions to contain the spread, and distinguish healthcare workers who have been infected, and are required to self-isolate, from those showing similar symptoms but which are not 2019-nCoV associated. The latter category may continue to work, alleviating stress on hard-pressed healthcare resources. 2019-nCoV is an RNA virus, and the diagnostic tests detect viral RNA in swabs from patient airways using a reverse transcriptase PCR assay. Samples are submitted to HSL, an accredited reporting laboratory, and transferred to the Crick for testing.Principles of ExaminationThis procedure involves the preparation of TET buffer for the RNA extraction protocol.
Proper citation: Nicola O'Reilly, Svend Kjaer, Maria Greco 2020. Preparation of TET Buffer. protocols.io dx.doi.org/10.17504/protocols.io.bfeujjew Copy
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