Tutorials

: Super User; Tutorials; 01 November 2022; Hits: 1140

What a student should read

Initiation literature for master and PhD students.

A sense of history:

First Nature article ever by Goethe (1869)

The 3 nature papers on DNA structure published in 1953: The Watson-Crick paper is one of the best written manuscripts I have ever read. Please read the delicate three lines on p. 558 by Watson & Crick starting with ("It has not escaped our notice.....", rarely has ONE sentence hidden so much depth and comprehension).

Guides:

How to become a scientist by Pr. Yewdell: First, Second

Microbiota terminology by Pr. Ravel.

Immunity - self versus non-self to the danger model: Matzinger et al. Scand J Immunology 2003

B cell immunology at the extremes of age: Blanco et al. JACI 2018

Shortest articles and thesis ever

Microbiota biology:

Whittaker's Ecology paper (alpha, beta and gamma diversity): Whittaker et al. Taxon 1972

Natural biotinylation of bacterial proteins (Streptavidin as a detection reagent): Matsuhisa et al. Microbiol Immunol 1993

Microbiota in numbers by Dr. Sender: Sender et al. PLoS Biology 2016

Specific literature for Immunity & Microbiota Ecology:

IgA regulatory mechanisms: Bunker et al. Immunity 2018 Review

Immune exclusion: Moor et al. Nature 2017

Immune inclusion: Donaldson et al. Science 2018

IgA immunity to microbes:

IgA binds colitogenic bacteria: Palm et al. Cell 2014

IgA bindings associated with infant malnutrition: Kau et al. STM 2015

IgA binds preferentially Small Intestine microbes: Bunker et al. Immunity 2015

IgA regulates microbiota ecology: Fadlallah et al. STM 2018

Microbial superantigens target immunoglobulin Fab region (C. comes, R. gnavus): Bunker et al. STM 2019

IgAseq analysis : Jackson et al. Microbiome 2021 and IgAscore R package

IgG immunity to microbes:

Seric IgG binds microbiota (flow cytometry): Slack et al. Science 2009

Seric IgG microbial specificity converge with gut IgA: Fadlallah et al. JACI 2019

Seric IgG responses to fungi: Moreno-Sabater et al. Microbiome 2020

Bacterial degradation of secretory component: Moon et al. Nature 2015

Immune memory and persistance (long-term effect of early-life exposure): Vergani et al. Immunity 2022 and Ramanan et al. Immunity 2020

Technical/experimental limitations in microbiota research:

Minimum 10⁷ microbes for robust WGS (solid): Plaza-Onate et al. BMC Genomics 2015

Minimum 10⁶ microbes for robust 16S rRNA gene analysis: VILLETTE et al. Sci Rep 2021

Practical experimental notes:

Safety

All laboratory staff NEED (obligatory) to have completed Inserm's Néo : accueil et prévention (expect 4x20min).

Always wear lab coat and gloves when experimenting.

Make sure to treat waste correctly. Ask your supervisor or colleagues if in doubt. A. Liquid waste should be marked with your initials, U1135, date and a description of the content (should be placed in the corridor on Wednesday afternoon). B. Solid waste should be marked with your initials, U1135 and the date.

Antibodies

Antibodies are playing a major role in our research. Both directly as our target of interest as well as indirectly, because they represent essential tools for the detection of various biomolecules (Flow cytometry, ELISA etc.). Antibody reagents used for research can be unconjugated or conjugated with biotin, HRP or various fluorochromes. They are generally very expensive (3-600 Euros per bottle/tube). HOW should you preserve these reagents.

1. Always work on ice (when you take the tube out of the fridge or freezer keep them on ice at all times).

2. Avoid freeze-thaw cycles. For antibodies that requires to be stored frozen make aliquotes. When aliquotes are thawn (only after verifying that an aliquote is not already available in fridge) do NOT re-freeze them (keep in fridge).

3. Antibodies are generally very stable and can be stored for quit a long time in fridge (follow manufactures recommendation)

4. For long-term storage antibodies can be frozen at -80dC (AVOID for PE conjugated antibodies). Make aliquotes of a reasonable size, which can be left in fridge after thawing for a few months (avoid freeze-thaw cycles). NEVER freeze a completely batch of antibody.

5. Rarely (only after discussing with your supervisor) antibodies can be frozen at -20dC, but in this case adding upto 50% glycerol is an advantage as it will benefit from the low temperature but avoid crystalization (even at -20dC the antibody solution is still liquid).

6. Samples containing antibodies (serum, fecal water, breastmilk etc.) are generally stored at -80dC (long-term storage), but if they have been diluted or plated out for experimental use with no need for long-term storage they can be stored at -20dC (please consult your supervisor).

Beyond the impact the above guidelines could have on the experimental quality of your work, antibodies are also a major part of our lab budget. Please take care of them.

Sequencing technology:

Metagenomic sequencing - principles:

Technical constraints:

Ion Torrent:

Illumina sequencing technology:

Oxford Nanopore technology (e.g. minION, PromethION)

Practical analytical notes:

R coding tutorials:

Riffomonas by Pat Schloss author of Mothur

Statistical software:

Free Graphprism-like statistical software (installs on all platforms)

BiostaTGV including Power analysis

Polyfunctionality / Poly-Ig analysis:

Polyfunctionality presentation

Exhaustive boolean gating can be done in Flow Jo using the "Combination gates" function. Exporting this data can then be analyzed with the FunkyCells software.

Polyfunctionality sample data (T cells)

Pubmed

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