-
What are some methods to examine genome wide expression? (6)
- FACS-RNAseq
- INTACT-RNAseq
- TRAP-RNAseq
- Laser Capture Microdissection-RNAseq
- Single Cell RNAseq
- Spatial Transcriptomics
-
FACS-seq
- Florescence Activated Cell Sorting
- specialized flow cytometry
- sorts a heterogenous mixture of biological cells, one cell at a time, based on specific light scattering and fluorescent characteristics
- Helps to assign biological function to genes of a specific cell type
-
INTACT-seq
- Isolation of Nuclei TAgged in specific Cell Types
- requires a transgenic line
- Nuclei is released, and GFP is targeted to the nuclear envelope
- beads attract the GFP protein and all you need is a magnet to pull them out of solution from the unbound cellular debris
- followup with RNA-seq
-
TRAP-seq
- Translating Ribosome Affinity Purification
- requires a transgenic line
- gentler than INTACT
- specifically for pulling down ribosomes only in the cells where the promoter is active
- make RNAseq libraries and sequence them to get information about the transcriptome
-
Laser Capture Microdissection
- does not require transgenic lines
- obtain tissue within desired cells and place beneath a specialized film.
- Laser hits film in places you are interested and melts cells onto the film
- Typical RNA seq experiments will use higher volumes of RNA than this provides, but can still be used in RNA seq experiments
-
Improvements on RNA seq that enable libraries to be created with only a few cells
- Tang's method: a single cell is picked and directly lysed, and mRNA is made into cDNA via oligo-dT primer. The resulting amplified cDNA. This method is adequate for constructing a library for NGS
- Smart: Improves efficiency by starting with more RNA. generates and amplifies full cDNA from a single cell using reverse transcriptase from M-MLV. Template switching and terminal transferase are critical to this method's success
- Islam's method: uses a UMI to ensure that your amplified transcript reflects the original transcripts
-
Islam's method for creating scRNA-seq libraries
- uses a UMI to ensure that your amplified transcript reflects the original transcripts
- overcomes errors and bias derived in RNA loss in cDNA synthesis
- adding UMIs nearly eliminates amplification noise
- multireads with the same UMI are regarded as one read in bioinformatics analysis
-
CEL-seq
- early method of scRNA-seq that brute forced one cell per well with a barcode for amplification and creation of their library
- makes use of wells
-
DROP-seq
- molecular barcoding of cellular transcriptomes in droplets
- tissue separated into individual cells, which are encapsulated in droplets with barcoded microprimers
- mRNA reverse transcribed into cDNA, generating a set of beads called "single-cell transcriptomes attached to microparticles (STAMPS)"
- STAMPS are then amplified and sequenced
-
InDrop
- Cells are encapsulated into droplets with a lysis buffer, reverse-transcription mix
- hydrocel microspheres cary barcoded primers
- cDNA in each droplet is tagged with the barcode during RT
- droplets are broken and all cell material is amplified before sequencing
-
10xGenomics on GemCode single-cell platform
- this technology enables 30 profiling of RNAs from thousands of single cells simultaneously
- Gems formed from combination of cells and gel beads.
- RT takes place in each gem, after which cDNA is pooled for amplification and library construction
-
Visium vs Xenium for performance on gene sensitivity, gene detection and transcript abundance
- Xenium offered greater per-gene sensitivity
- Visium exhibited higher gene detection and greater transcript abundance
-
Sci-RNA-seq
- first does RT in the cell, bypassing the need for microfluidic sac for cDNA and amplification
- fix cells with methanol, put in wells
- cells are pooled and distributed into wells, and barcoded to ID per well
- cells are pooled again, distributed and barcoded
- repeated several times until each cell has a unique barcode
-
SPLiT-seq
- labels transcriptomes with split-pool barcoding
- fixed cells/nuclei distributed into wells
- first barcode delivered by barcoded RT primers
- 2nd and 3rd primers appended to cDNA via ligation
- 4th barcode added by PCR during library prep
-
What is a limitation of RNA-seq in single cell analysis?
- We only can get a few reads per cell
- we don't have the power to describe transcription rate
-
Experimental methods to preform multimodal single cell analysis
- gather single cell measurements using FACS before scRNA-seq then fluorescence based measurements of protein levels can be linked to cellular transcriptomes
- lyse-and-split strategy allows parallel workflow on different cellular fractions
-
10xGenomics: Visium
- a slide has probes mounted to it. Barcodes on the capture area give location information about tissue that is placed on the slide.
- Tissue is permeated and RNA binds to probes
- bound genetic data contains index barcodes UMIs and cDNA to be sequenced, giving spatial information about cellular transcripts
-
10xGenomics:Xenium
- in-situÂ
- probes look like padlocks and are very specific to target sequences, ligated and amplified by rolling circle amplification
- florescent barcodes bind to the tags and through successive imaging rounds, an optical signature identifies the transcripts
|
|
