Lineage tracing & bioinformatics | Quizlet

Lineage tracing methods

Dye-mediated labelling techniques, genetic markers, genetic fate mapping techniques, mosaic analysis

Dye-mediated labelling techniques

DiI/DiO, BrdU incorperation, HRP and fluorescent dextrans

DiI/DiO

Introduce lipophilic fluorescent carbocyanine dye molecule which binds to the cell membrane due to their lipophilic nature

BrdU incorperation

Thymidine DNA analogue is build into DNA and automatically gets replicated during cell division

When is BrdU incorperation often used?

Birthdating because it only shows fluorescence in cells that have proliferated

Benefit of using BrdU incorperation

You can play with the time window of introduction and the concentration to determine how long the signal is present

HRP and fluorescent dextrans

Add the dye directly to the cells, but the intensity of the dye halves every cell division

Different methods of introducing genetic marker during lineage tracing

Transfection, electroporation, viral infection

Transfection introduction of genetic markers

Use patch clamps, similar to the ones used to measure voltage and current, to introduce plasmids containing labelled RNA and isolating the RNA after time to sequence

Electroporation introduction of genetic markers

Shock cells to insert fluorescent dyes (GFP) in vivo

Viral infection to introduce genetic markers

Use adino virus labelled with GFP to build itsel into the DNA using TFs

Tropism

Specificity of adeno virus towards host cells, there are 7 tropism/affinity types towards neurons

Genetic fate mapping technique used for lineage tracing

Cre/Lox system

Cre/Lox system

Uses a Cre recombinase driver and a Lox(P) floxed sit, Cre is build into the genes of interest, while Lox(P) is build into all cells in the household genes, when both Cre and Lox(P) are present the reporter gene on the lox gene is expressed

Example of household gene used for Lox(P)

ROSA which is expressed in almost all cells

Problem with transgenesis systems like cre/lox

Can destroy the genome when it is built into regions that are not of interest, each replication the genome can change leading to the need to verify if transgenesis was done correctly

Multisystonic messenger

Messenger for multiple genes

Third enzyme that can be used in a cre/lox system

Flipase recombinase which can flip lox genes to make it recognisable for Cre, flipping can be used to switch on/off the genes

FACS

Fluorescent activated cell sorting

How can FACS be used in combination with Cre/Lox

Cells with activated genes of interest can activate transcription of fluorescent Lox genes, the cells can then be isolated (for scRNAseq)

Mosaic analysis

Intersectional fate mapping, further developed cre/lox-like system where different fluorescent dyes are built into multiple genes to look into expression patterns

Backend

The code language

Frontend

The ui needed to write and visualize the code

Gene expression omnibus (GEO)

Frontend for RNA transcripts, can reuse old data (microarray data), has raw and processed data

Accession codes

Unique identifiers to download SRR files with FASTq transcripts

How many reads does a FASTq file need

10^7

Why are there so many reads needed in a FASTq file?

Only a part of the total RNA is mRNA needed for PCR

How is the amount of mRNA increased for PCR

Selected using the poly-A tail, which can be filtered using poly-T probes

Single-end sequencing

PCR scanning done from one side of the DNA/RNA fragments

Paired-end reading

Software to speed up high throughput reads

What are good reads during quality control

The first few and last few nucleotides are often not good, sometimes left out if the reads are too problematic

What is wanted during sequencing

Unique sequences

HISAT2 and BLAST

Methods to map sequencing data to genome or other sequences, is fast, sensitive and can recognize spliced reads and bridges the intron gaps to look into the mapping on the exons only

Why is 100% alignment not possible

Mutations and differences between cell lines due to long time differences between isolated cells from 30 years ago

DESeq2 and LIMMA

Need n>2 for statistical analysis and to find differences between the mapped sequences

Public databases

GEO, UCSC, NCBI, Ensemble

How to work with scRNAseq data

There are a lot of files (~1600), use of datasets that has all the files is faster and more efficient

How to work with sequencing data from pharma

Difficult to read, so to perform analysis on large datasets, it is better to convert it into a matrix with R, same for feature count, it is better to convert is into a matrix to see differences

Western blot

Identifies proteins based on size

Southern blot

Divides DNA fragments based on size

South-western blot

Shows protein-DNA interaction based no size

Difference between conservation of introns and exons

Introns are less conserved due to the different promoters and transcription factors needed to bind as well as that introns can be spliced out, exons are more conserved

Why is ChIP-seq qualitative and not quantitative

The amount of DNA bound to proteins can differ greatly between cells

How can ChIP-seq be used quantitatively?

Compare DNA between different groups to look into differences in activity between active DNA

Cut&Run

Similar to ChIP-seq but measures less DNA but has higher specificity, difference is the enzyme which can penetrate the nucleus and therefore not requiring DNA isolation

Cut&tag

Similar to cut&run, but has Tn5 enzyme that can label the fragmented DNA to make the process more efficient

DAM-ID

Uses methylase to bind DNA and methyl groups are added to the DNA to label fragments, very similar to cut&tag