sc rna seq workflow

2025. 1. 1. 11:05Lab skills

Performing basic scRNA-seq


1. Separating live single cells from tissues of interest

2. Capture as much RNA as possible by lysing isolated cells - usually using poly[T]-primer

3. Transform poly[T]-primed RNA into cDNA with reverse transcriptase (adding adapter sequences for sequencing platforms used and barcoding tasks for cell origin - adding unique molecular identifieres (UMIs))

4. cDNA amplification via PCR or in vivo transcrption

5. Pooled amplified or tagged cDMA in all cells and sequenced with NGS





Kit types used in the scRNA-seq protocol of the Wet-lab stage:

- 'switching mechanism at 5' end of RNA template' (SMARTer) chemistry (Clontech Laboratories) for mRNA capture, reverse transcrption, cDNA amplification.

- Illumina's Nextera kits for barcoding cDNA libraries

- Droplet-based platforms: Chromium from 10x Genomics, ddSEQ from Bio-Rad Laboratories, InDrop from 1CellBio, μEncapsulator from Dolomite Bio/Blacktrace Holdings (These instruments can put thousands of single cells in each part and contain all reagents necessary for the wet-lab stage, omitting the flow-cytometric sotring and micro-dissection processes required for single-cell separation)

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