This protocol is for four time-points in 12-well plates. Scale up or down as needed.

Day 1:
Plate cells:
Split one or more 10-cm plate(s) with 80-100% confluent HeLa Tet-off cells to a final volume of 10-11 ml. Make sure to break up cell clusters to ensure single cells.
Dilute freshly split cell suspension 1:12 or 1:15 (depending on % confluency) in DMEM/10% FBS in a 50 ml conical in a total volume equal to or more than the number of wells to be plated (for e.g. make about 50 ml for 48 wells -12 experiments of 4 time-points).
Plate 1 ml per well in 12-well plates.
Incubate over night.

Day 2:
Transfections:
Using Mirus TransIT HeLaMonster (check manufacturers protocols if using other reagents)
Cells should be 40-60% confluent - not more.

• For each of four wells:

  Add a total of 4.5 µg plasmid to an eppendorf tube:
  For B-globin reporters use:
  45 ng pcBwtGAP3 UAC (internal control mRNA)
  0.9 µg pPC or pcTET2 B-globin reporter
  3.6 µg of other plasmid(s) - such as protein expressing or empty vectors

• Optional: If using same reporters in all experiments, make a larger mix of internal control and B-globin reporter. For e.g., for 12 experiments, mix

  45 ng x 12.5 of pcBwtGAP3 UAC (internal control mRNA)
  0.9 µg x 12.5 of pPC or pcTET2 B-globin reporter
  Then, divide the mix into 12.5 equal parts and pipet into separate eppendorfs.

• Mix 225 µl O-MEM with 13.5 µl TransIT HeLa reagent

  Add 0.225 µl of 1 mg/ml Tetracycline (in EtOH).
  (a final concentration of 50 ng/ml Tet ensures no expression from the TET-driven promoter)
  Incubate 5-20 mins at RT

• Mix 5 µl HeLaMonster reagent with 45 µl ddw. Store on ice.

  (Important: Thaw HeLaMonster reagent just before use, keep on ice and return to freezer immediately).
  Optional: Bigger mixes can be made by multiplying O-MEM, TransIT reagent and Tetracycline volumes (Step 2) or HeLaMonster and ddw volumes (Step 3) by the number of experiments (+0.5).

• Add 225 µl O-MEM/TransIT HeLa mix to the plasmid mix.
  >Incubate 5-20 mins at RT
• To each 2.0-cm well of HeLa Tet-off cells:
  Add 50 µl of O-MEM/TransIT/Plasmid mix.
• To each 2.0-cm well of HeLa Tet-off cells:

  Add 10 µl of diluted HeLaMonster reagent.
  Place plates in incubator.

Day 4:
Pulse-Chase assay.
- Pulse transcription (Early morning):

Start transcription from Tet-promoter:
Wash wells carefully (!) with 1 ml PBS.
Add 1 ml DMEM/10% FBS.
Incubate ≈6 hours
- Chase:
Stop transcription (the complete stop will take ≈20 mins):
To each well, add 10 µl of O-MEM/100 µg/ml Tetracycline
Place back in incubator.
- Time points:
Take the first time point 20-30 mins after addition of tetracycline (t=0).
For each time point:
Wash cells with 1 ml PBS.
Add 0.5 ml Trizol. Pipet up and down until non-viscous.
Transfer to pre-labeled eppendorf tube.
Store in -20 C.

Day 5:
RNA preps:
To each tube with 0.5 ml Trizol:
Add 100 µl Chloroform.
Mix by shaking for 30 sec or more.
Leave at RT, 10 min.
Spin 12,000 rpm, ≈10 min.
Transfer 250 µl supernatant to new tube. Avoid interphase!!
Add 0.7 volumes of isopropanol (175 µl). Mix and leave at RT, 10 min.
Spin 12,000 g (not faster!), 4C, 10 min.
Remove supernatant carefully.
Wash carefully with 0.5 ml of 70% EtOH. Spin at 7,500 g for 5 min, 4C.
Air dry.
Dissolve in 10 µl formamide load buffer.
Run 5 µl on a Northern Gel.