Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2016/08/02

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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) Nothing new. 2. Dox dose curve followed by flow cytometry and qRT-PCR on gene panel (subset of 8 genes + GAPDH + PcTF); also do this for TF cell line if possible Performing cDNA synthesis on samples 1.1-1.3. Following instructions located here. Each sample will be reacted in triplicate (60 µL total volume). Sample ID ng/µL 260/280 1.1 39.122 1.956 1.2 52.618 1.953 1.3 35.561 1.933 oligo(dT) Primer-RNA annealing reactions Reagent Vol total RNA (up to 2μg) up to 8 μL 50 μM oligo(dT) primer 1.0 10 mM dNTP mix 1.0 Water (SS III kit) = 8.0 - vol. total RNA   10.0 μL --> Incubate at 65°C/ 5 min. Immediately place on ice for 1 min. cDNA synthesis mix Total reactions = 9 Samples: 1.1 rxn 1 1.1 rxn 2 1.1 rxn 3 1.2 rxn 1 1.2 rxn 2 1.2 rxn 3 1.3 rxn 1 1.3 rxn 2 1.3 rxn 3 Reagent Single rxn. Mix (x10) 10x RT buffer 2.0 20 25 mM MgCl2 4.0 40 0.1 M DDT 2.0 20 RNaseOUT 1.0 10 SuperScript III RT 1.0 10   10.0 μL 100 μL --> Aliquot 10 μL of mix into 8-tube strip --> Add annealing rxn. into each 10 μL aliquot --> PCR machine: 50°C/ 50 min., 80°C/ 5 min., 4°C/ ∞ --> Add 1.0 μL RNase H, incubate at 37°C/ 20 min. --> Store at -20°C Meanwhile, start a new set of two 6-well plates of U2OS-PcTF and when sufficiently high cell density, induce with dox at 1 µg/mL, 31 ng/mL, 16 ng/mL, and 0 ng/mL (control) in triplicate (12 wells total) for 4 days. This will give us enough material for a higher-yield RNA extraction next week if needed. 3. qRT-PCR experiments with TF control plasmid, on U-2 OS, SK-N-SH and K562 cell lines. Cannot select stables because PcTF is toxic. Try normalizing to PcTF levels. Include CDKN2A. Performing cDNA synthesis on U2OS-KAH132 transfected cells. Following instructions located here. For 72 hour samples, use 7.1 µL of RNA for sample 1 and 5.9 µL of RNA for sample 2. Sample ID ng/µL 260/280 U2OS KAH132 24hr rep1 117.411 1.932 U2OS KAH132 24hr rep2 137.561 1.926 U2OS KAH132 48hr rep1 146.72 2.045 U2OS KAH132 48hr rep2 119.495 2.006 U2OS KAH132 72hr rep1 280.054 2.026 U2OS KAH132 72hr rep2 339.671 2.048 oligo(dT) Primer-RNA annealing reactions Reagent Vol total RNA (up to 2μg) up to 8 μL 50 μM oligo(dT) primer 1.0 10 mM dNTP mix 1.0 Water (SS III kit) = 8.0 - vol. total RNA   10.0 μL --> Incubate at 65°C/ 5 min. Immediately place on ice for 1 min. cDNA synthesis mix Total reactions = 9 Samples: U2OS KAH132 24hr rep1 U2OS KAH132 24hr rep2 U2OS KAH132 48hr rep1 U2OS KAH132 48hr rep2 U2OS KAH132 72hr rep1 U2OS KAH132 72hr rep2 Reagent Single rxn. Mix (x7) 10x RT buffer 2.0 14 25 mM MgCl2 4.0 28 0.1 M DDT 2.0 14 RNaseOUT 1.0 7 SuperScript III RT 1.0 7   10.0 μL 70 μL --> Aliquot 10 μL of mix into 8-tube strip --> Add annealing rxn. into each 10 μL aliquot --> PCR machine: 50°C/ 50 min., 80°C/ 5 min., 4°C/ ∞ --> Add 1.0 μL RNase H, incubate at 37°C/ 20 min. --> Store at -20°C 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) Samples submitted to DNASU on 7/29.