Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2016/07/24

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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)

Update: Dr. Haynes has ordered primers (check this). Cell lines are being split into plates by Rene and Ben. Looks like the U2OS-TF line is not growing.

To do: Check U2OS-PcTF cell line and split into T-75 for Rene.

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

Update: KAH126-U2OS cells were split into two 12-well plates. Dox induction began at 5pm today. Will harvest cells for flow cytometry, RNA extraction and cDNA synthesis on 7/28.

Order more of the RNA extraction kit.

Dox induction plan:

  • S1: 1 µg/mL
  • S2: 0.5 µg/mL
  • S3: 0.25 µg/mL
  • S4: 0.125 µg/mL
  • S5: 63 ng/mL
  • S6: 31 ng/mL
  • S7: 16 ng/mL
  • S8: no treatment control

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.

Update: don't know status of U2OS cells, need to check on that. Also need to make more KAH132_MV2 plasmid. Transforming DH5α-T cells overnight, will start liquid culture tomorrow afternoon.

Check for levels of probes, order more if needed.

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)

Update: KAH126-U2OS cells were induced on Friday, and U-2OS cells were transfected with KAH132 on Friday. Visible expression in both plates (see below).

KAH126-U2OS, 48 hours post induction.

Well 1:
2016-07-24 Well 1 1 ug-mL doxycyclin phase mCherry 10x.jpg

Well 2:
2016-07-24 Well 2 1 ug-mL doxycyclin phase mCherry 10x.jpg

Well 3:
2016-07-24 Well 3 1 ug-mL doxycyclin phase mCherry 10x.jpg

U-2 OS transfected with KAH132_MV2, 48 hours post transfection.

Well 1:
2016-07-24 Well 1 KAH132-MV2 phase mCherry 10x.jpg

Well 2:
2016-07-24 Well 2 KAH132-MV2 phase mCherry 10x.jpg

Well 3:
2016-07-24 Well 3 KAH132-MV2 phase mCherry 10x.jpg

Will harvest two KAH126 +dox wells, two KAH126 no dox wells, and two U-2OS + KAH132-MV2 transfection wells on 7/26. Will also take photos prior to harvesting for RNA-seq.