Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/06/10

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Overview

Day 2 of collecting flow cytometry data, fluorescence microscopy data, and harvesting cells for RNA.

Also need to set up two more samples for a "no plasmid" control transfection. Current cell density of K562 culture is 420,000 cells/mL. Need to wait until culture is around 800,000 cells/mL before proceeding with mock transfection.

Following the protocol for RNA miniprep found here.

Fluorescence Microscopy

Well 4: pMaxGFP, post-48hr

2015-06-10 Well 4 pMaxGFP phase GFP 10x.jpg

Well 5: KAH126-MV2, post-48hr

2015-06-10 Well 5 KAH126-MV2 phase mCherry 10x.jpg

Well 6: KAH126-MV2, post-48hr

2015-06-10 Well 6 KAH126-MV2 phase mCherry 10x.jpg

Flow Cytometry

Plot 1 (FSC-A/SSC-A) Plot 4 (FL3-A) Plot 6 (FL1-A)
All Cells M1 M2
Count Volume (µL) Events / µL Count Events / µL % of All Count % of This Plot Count % of This Plot
A02 0 untreated 3,967 10 397 2,930 293 73.86% 13 0.44% 15 0.51%
A06 4 pMaxGFP 21,814 10 2181 10,666 1067 48.90% 6,378 59.80% 9,945 93.24%
A07 5 KAH126-MV2 11,155 10 1116 5,547 555 49.73% 674 12.15% 368 6.63%
A08 6 KAH126-MV2 8,363 10 836 3,636 364 43.48% 330 9.08% 24 0.66%
A09 0 untreated 3,881 10 388 2,862 286 73.74% 2 0.07% 1 0.03%

RNeasy / Trizol

Samples are stored at -80 °C.