Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/01/23: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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==Summary== | ==Summary== | ||
The KAH126/U2OS cells in the 6-well plate that were induced to produce PcTF-RFP back on Monday are ready for the first round of harvesting. I'll be isolating the RNA from them and storing the RNA at -80°C before sending the samples out for analysis early next week. | |||
To be clear, I currently have two batches of KAH126/U2OS induced by doxycycline. One batch was inoculated on 1/16 and induced 1/19. I'm going to be harvesting three of the wells from that batch for RNA sequencing today. The other batch was inoculated on 1/20 and induced 1/22. I've taken fluorescence microscopy photos of both experiments. | |||
Also need to passage the SKNSH culture from PS-β to PS-1 and the KAH126/U2OS culture from PS-4 to PS-5. | |||
<br><br> | <br><br> | ||
==Goals== | ==Goals== | ||
* | * Passage SKNSH | ||
* Passage KAH126/U2OS, save some for Karmella's plates | |||
* Harvest RNA from KAH126/U2OS induced on Jan 19th | |||
<br><br> | <br><br> | ||
==Protocol== | ==Protocol== | ||
[http://openwetware.org/wiki/Haynes: | [http://openwetware.org/wiki/Haynes:TRIzol_RNeasy TRIzol/RNEasy Spin Column Protocol] | ||
[http://openwetware.org/wiki/Haynes:SplittingCells passaging mammalian cells] | |||
<br><br> | <br><br> | ||
==Notes== | ==Notes== | ||
Observations, measurements, placeholder info etc. go here. | Observations, measurements, placeholder info etc. go here. | ||
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==Results/Conclusions== | ==Results/Conclusions== | ||
Photographs of the KAH126/U2OS samples induced on 1/19 (4 days post-induction): | |||
[[Image:2015-01-23_KAH126-U2OS_day4postinduction_Well_1_phase_%26_mCherry_10x.jpg | 250px | Well 1]] [[Image:2015-01-23_KAH126-U2OS_day4postinduction_Well_2_phase_%26_mCherry_10x.jpg | 250px | Well 2]] | |||
[[Image:2015-01-23_KAH126-U2OS_day4postinduction_Well_3_phase_%26_mCherry_10x.jpg | 250px | Well 3]] [[Image:2015-01-23_KAH126-U2OS_day4postinduction_Well_4_phase_%26_mCherry_10x.jpg | 250px | Well 4]] | |||
[[Image:2015-01-23_KAH126-U2OS_day4postinduction_Well_5_negative_control_phase_%26_mCherry_10x.jpg | 250px | Well 5 (negative control)]] [[Image:2015-01-23_KAH126-U2OS_day4postinduction_Well_6_negative_control_phase_%26_mCherry_10x.jpg | 250px | Well 6 (negative control)]] | |||
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Photographs of the KAH126/U2OS samples induced on 1/22 (1 day post-induction): | |||
[[Image:2015-01-23_KAH126-U2OS_day1postinduction_Well_1_negative_control_phase_%26_mCherry_10x.jpg | 250px | Well 1 (negative control)]] [[Image:2015-01-23_KAH126-U2OS_day1postinduction_Well_2_negative_control_phase_%26_mCherry_10x.jpg | 250px | Well 2 (negative control)]] | |||
[[Image:2015-01-23_KAH126-U2OS_day1postinduction_Well_3_phase_%26_mCherry_10x.jpg | 250px | Well 3]] [[Image:2015-01-23_KAH126-U2OS_day1postinduction_Well_4_phase_%26_mCherry_10x.jpg | 250px | Well 4]] | |||
[[Image:2015-01-23_KAH126-U2OS_day1postinduction_Well_5_phase_%26_mCherry_10x.jpg | 250px | Well 5]] [[Image:2015-01-23_KAH126-U2OS_day1postinduction_Well_6_phase_%26_mCherry_10x.jpg | 250px | Well 6]] | |||
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Photographs of the U2OS samples transfected on 1/22 (1 day post-transfection): | |||
[[Image:2015-01-23_U2OS_1dayposttransfection_Well_1_phase_%26_mCherry_10x.jpg | 250px | Well 1]] [[Image:2015-01-23_U2OS_1dayposttransfection_Well_2_phase_%26_mCherry_10x.jpg | 250px | Well 2]] | |||
[[Image:2015-01-23_U2OS_1dayposttransfection_Well_3_phase_%26_mCherry_10x.jpg | 250px | Well 3]] [[Image:2015-01-23_U2OS_1dayposttransfection_Well_4_phase_%26_mCherry_10x.jpg | 250px | Well 4]] | |||
Wells 3 and 4 were transfected with a plasmid made from a different batch than wells 1 & 2, thus the greatly reduced efficiency. | |||
[[Image:2015-01-23_U2OS_1dayposttransfection_Well_5_negative_control_phase_%26_mCherry_10x.jpg | 250px | Well 5 (negative control)]] [[Image:2015-01-23_U2OS_1dayposttransfection_Well_6_negative_control_phase_%26_mCherry_10x.jpg | 250px | Well 6 (negative control)]] | |||
----- | |||
RNA extraction of wells 1, 2 and 4 from the KAH126/U2OS plate induced on 1/19 (first set of images, above) was successful. RNA is in the -80°C freezer awaiting analysis. | |||
Latest revision as of 00:40, 27 September 2017
 RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines
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 Main project page Previous entry Next entry
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SummaryThe KAH126/U2OS cells in the 6-well plate that were induced to produce PcTF-RFP back on Monday are ready for the first round of harvesting. I'll be isolating the RNA from them and storing the RNA at -80°C before sending the samples out for analysis early next week. To be clear, I currently have two batches of KAH126/U2OS induced by doxycycline. One batch was inoculated on 1/16 and induced 1/19. I'm going to be harvesting three of the wells from that batch for RNA sequencing today. The other batch was inoculated on 1/20 and induced 1/22. I've taken fluorescence microscopy photos of both experiments. Also need to passage the SKNSH culture from PS-β to PS-1 and the KAH126/U2OS culture from PS-4 to PS-5.
Goals
ProtocolTRIzol/RNEasy Spin Column Protocol
NotesObservations, measurements, placeholder info etc. go here.
Results/ConclusionsPhotographs of the KAH126/U2OS samples induced on 1/19 (4 days post-induction):
Photographs of the KAH126/U2OS samples induced on 1/22 (1 day post-induction):
Photographs of the U2OS samples transfected on 1/22 (1 day post-transfection):
Wells 3 and 4 were transfected with a plasmid made from a different batch than wells 1 & 2, thus the greatly reduced efficiency.
RNA extraction of wells 1, 2 and 4 from the KAH126/U2OS plate induced on 1/19 (first set of images, above) was successful. RNA is in the -80°C freezer awaiting analysis.
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