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Old 01-05-2018, 09:11 AM   #1
alekzs
Junior Member
 
Location: CT

Join Date: Jan 2018
Posts: 8
Default Single cell RNA-seq trouble

Hi folks,
I'm trying to set up a single-cell RNAseq platform for human primary cells, so far semi-successfull. Hopefully you can give me some feedback on how to optimize and fix the problems.

Here's what I do, somewhat following the Trombetta protocol http://onlinelibrary.wiley.com/doi/1...2s107/abstract :
1) FACS sort 1 cell/well into PCR plate with 10ul TCL buffer (+bMerc)
2) 2.2x RNA SPRI
3) RT with Maxima RNaseH-minus RT, 90min at 52C, then 10cycles 2min-50C, 2min-42C
4) WTA with KAPA HiFi Hotstart, 20 cycles
5) 2x 0.8x DNA SPRI

The good: I tested the protocol with Jurkat cells and got acceptable BioA traces for both a bulk control as well as 1cell/well samples (see attachment).

The bad: Using primary cells (human effector T cells or regulatory T cells, with the Tregs being my goal population) it went south. It works with >200 cells but I either get nothing out of my single cells or a weird pattern of cDNA distribution with 2-3 small bumps (I wouldn't even call them peaks).

What can I try to get it to work with 1 primary cell? And what causes this weird bumpy pattern?

Thanks
Alex
Attached Files
File Type: pdf scRNAseqtrouble0118.pdf (376.9 KB, 43 views)
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