Genetics and Molecular Biology

Lectures 1-4 (Prof.ssa Morlando) - Introduction to the complexity of the RNA world - Methods for generation of cellular model systems for the study of cell differentiation and tissue organization. Examples of 2D in vitro muscular and neuronal differentiation as well as generation of 3D cultures (e.g. organoids). - Different applications of CRISPR technology: a) to perform genome editing; b) to reversibly control gene expression; c) to perform genetic screen Lectures 5-7 (Prof. Setti) - practical methodologies for the design of CRISPR-KO experiment combined with the use of RNA-sequencing to explore the effect of the KO on gene expression. The lesson will include RNA-sequencing analysis using Galaxy platform to perform FASTQ files preprocessing and alignment to reference genome. IGV software will be used to explore the deletions or mutations induced in the target locus in the cells through the analysis of the aligned reads. Then, the gene expression quantification, differential expression analysis and GO term enrichment analysis will be presented Lectures 8-9 (Prof.ssa Morlando) - Single cell approaches droplet- and in wells based - Spatial transcriptomics Lectures 10-12 (Prof. Setti) - Methods for analysis of single-cell transcriptomic data from different model systems, including: data processing, cells quality filtering, dimensionality reduction and clustering , basic integration methods, differential expression methodologies to identify cell population identity or to compare conditions (e.g. WT vs KO), trajectory analysis using Monocle or RNA-velocity. This module will include in-depth analysis of selected scientific papers used as reference (Immune cell system example, Neuro-muscolar organoids etc.) - Methods for integrating multi-omic data in single cell omics, such as epigenome (ATAC-seq), transcriptome (RNA-seq) and genomic barcodes. Pathway analysis and Module-score calculation. Advanced methodologies, such as Topic modelling and Scenic+ application will be mentioned. Lecture 13 (Prof. Setti) RNA biology investigation using web tools Including, i) web tool used for investigating the expression of genes in various organisms, tissues and pathological conditions (i.e. GTEX), ii) investigating genetic dependencies using large-scale functional characterization projects i.e. DEPMAP) and cancer datasets (i.e. CBIO portal). iii) Database Spatial Transcriptomics applied to human brain. Lectures 14-15 (Prof.ssa Morlando) Short RNA (microRNA): - Short RNA sequencing methods to study microRNA Expression, - Methods for the study of their mode of action and target identification (i.e CLASH). - Methods for inhibiting endogenous expressed miRNAs or for rescuing their lost expression in pathological condition. - MiRNA in body fluids: origin and use as biomarkers; methods for their detection. Lectures 16-17 (Prof. Setti) - Introduction to chimeric-reads. In depth explanation of CLASH methodology that allows to capture bound microRNAs including the RNA elements targeted by the microRNAs. - Characterization of RNA:RNA interactions with different approaches based on psoralen derivatives including PARIS, SPLASH and COMRADES, protein-based approaches including MARIO and RIC-seq or new frontiers such as kethoxal-based methodologies (KARR-seq). RNA-SPRITE approach. Lectures 18 (Prof. Setti) - Introduction to RNA secondary structure. -Analysis of the main approaches used to probe RNA structures. Comparison of SHAPE and DMA-Map-seq. Lectures 19-21 (Prof.ssa Morlando) - New concepts in transcriptomics: epitranscriptomics. The importance of the epitranscriptome in controlling gene expression and in contributing to different disease phenotypes. - Innovative methods for capturing RNA Sequence/modification and Transcript Diversity. - Long reads sequencing: Oxford Nanopore and PacBio Lecture 22 (Prof.ssa Morlando) - Principles of epitranscriptomic analysis, available tools, comparisons, and example of one workflow (i.e. Nanocompore). - Practical session with detailed approach to data analysis from native RNA sequencing experiments Lectures 23-24 (Prof. Setti) - Introduction to RBPs functions and regulatory activities. - Methodologies to study RBP binding sites (RNAcompete, CLIP methodologies, etc.). - Methodologies used to study RNA-modifications sites in RNA: examples of meRIP-seq and mi-CLIP.

A biological or biomedical background is required, including basic knowledge of molecular biology, biochemistry and cellular biology. Familiarity with reading scientific journals in English is useful but not necessary. For the “Analysis Tools” module, no previous experience is necessary, but familiarity with the internet and basic operating systems (Linux, Mac OSX, Windows 10) is preferable.

e-learning (scientific articles and materials provided by the professor)

Attendance is not mandatory but strongly recommended.

The assessment method will consist of an oral examination where the student is invited to present a scientific article of their own interest in which one or more methodologies covered during the course are applied. This will be followed by a discussion and questions extended to all topics of the program.

The lessons will be conducted as lectures with the presentation of slides, which will subsequently be made available to the students. During the lectures, discussion on the different topics in the program will be encouraged in order to assess the student's degree of acquisition of the covered topics lesson by lesson.