Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2016/07/22

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Overview

Submitted paper to Nature Biotech. Reviewers returned it with revisions. Going to outline what needs to be done and create a roadmap to do everything.

Link to revision strategy: https://docs.google.com/document/d/1n-SZWgOC5y5N8kmKuQK9YiI6YXLkj5TkLs3aLen4HuI/edit

I'm only going to include the aims that I'm in charge of; other lab members are working on their own sections.


Major Aims

1. U2OS-PcTF and U2OS-TF stable cell lines ChIP-PCR on (1) PcTF, (2) H3K27me3, (3) H3K4me3, and (4) PolII before, during, and after PcTF expression at endogenous enhancer(s)

Goal: We want to see if PcTF actively changes chromatin state (and marks) while it's expressed, or if the H3K27me3 marks remain while PcTF upregulates by recruitment of Polymerase II.

Process: Design PCR primers (Haynes is working on this). Thaw and expand KAH126-4-U2OS and KAH132-8-U2OS. Split into 6 well plates. 3 plates per sample, 2 cell types per treatment. We want to harvest cells for ChIP immediately before induction with doxycyclin, during induction (how many days in?), and after induction (how many days after, and should we passage in the mean time?). Going to be a total of 18 wells. Need to find out how to prep cells for ChIP-PCR.


2. Dox dose curve followed by flow cytometry and qRT-PCR on gene panel (subset); also do this for TF cell line if possible

Goal: see if dox response is gradual or sudden, test robustness of system.

Process: Select 7 dox concentrations (including no dox control). For each concentration, set up 3 wells for each of KAH126-U2OS and KAH132-U2OS cell lines (6 wells per concentration, 42 wells total). When at sufficient density, induce expression of PcTF/delta-TF by addition of dox. Let grow for a couple days (how long?) and then harvest RNA for qPCR. qPCR 16-gene panel.


3. qRT-PCR experiments with TF control plasmid. Cannot select stables because PcTF is toxic. Try normalizing to PcTF levels. Include CDKN2A.

Goal: Using another control for the K562, SK-N-SH and U-2 OS cell lines. Transient transfection with TF plasmid to demonstrate that upregulation of genes isn't an artifact of higher TF present in cells.

Process: Thaw K562, SKNSH, U2OS. Grow in 6 well plate format. Transient transfection using KAH132. Harvest RNA for qPCR.


4. Another round of RNA-seq: (1, 2) U2OS-PcTF cells no-dox, (3, 4) U2OS-PcTF cells +dox ...and for good measure (5,6) U-2OS delta-TF (transient)

Goal: a repeat of KAH126-U2OS RNA-seq data, so we can have it in triplicate. Also want to do a triplicate RNA-seq of transiently transfect KAH132-U2OS.

Process: Grow up KAH126-U2OS, induce with dox (4 days), harvest for RNA-seq (in duplicate +1 extra). Also grow up KAH126-U2OS and don't induce with dox, harvest for RNA-seq. Do the same for U-2 OS, transiently transfect with KAH132.




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