Flow sorting | single cell | 单细胞 | Perturb-seq | CRISPR screen

流式细胞仪的cell sorting是很多细胞和分子生物实验的基础，比如单细胞测序、细胞周期等。

Flow cytometry and cell sorting are two distinct yet complementary techniques.

Both rely on antibodies to detect specific cells within a heterogeneous population, but while flow cytometry measures the proportion of each cell type, cell sorting does more. Based on the cytometry measurements, cell sorters isolate one or more cell populations from the pool. As demand for single-cell technologies continues to grow, flow cytometers and cell sorters are evolving to meet researchers’ needs.

常见筛选一：FSC-A & SSC-A

Forward versus side scatter (FSC vs SSC) gating is commonly used to identify cells of interest based on size and granularity (complexity).

It is often suggested that forward scatter indicates cell size whereas side scatter relates to the complexity or granularity of the cell.

常见的用于筛选完整单细胞，左边是debris，中间是single cells，右边是doublets。

选取中间部分进行下一级分选。

常见筛选二：荧光标记（mKate or GFP）& SSC-A

筛选特定的细胞，比如用mKate标记的有gRNA的细胞，或者GFP标记的有SOX9表达的细胞。

通常还需要negative control，比如筛选有gRNA的mKate，就用没有任何gRNA的细胞做对照。比如Perturb-seq实验。

也可以不用对照，比如CRISPR screen实验，只需要对比TOP 10%和BOTTOM 10%的细胞即可，然后collect genome DNA，amplify gRNA，NGS。

Perturb-seq需要high titer concentration这一步，富集lentivirus（10^8 virus/ml，qPCR可以准确测定），然后做titration滴定，确定最佳的病毒浓度（不是越高越好），通常选择能侵染30%的那个病毒浓度，这是大部分细胞只有一个gRNA。

常见筛选三：DAPI for live cells

DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent stain often used to differentiate between live and dead cells for viability.

活细胞是无法被DAPI侵染的。

CRISPR screen

pCC_01 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-Cas9-NLS-2A-Puro-WPRE (Plasmid #139086)

Perturb-seq

pCC_01 and pCC_09 (Addgene #139094) were modified to incorporate Capture Sequences 1 and 2 at the 3’ end of guide sequences. Oligo cloning and library preparation were done as described above. The Capture Sequences allowed sgRNAs to be captured within the same cell when cells were processed according to the Chromium Single Cell 3’ Reagent Kits v3.1 with Feature Barcoding technology for CRISPR screening protocol (10X Genomics, CG000205).