LaCava Research Wiki

AIM/HYPOTHESIS

See Endogenous a-ORF1 RNA-seq_Lars.

This experiment was performed together with Hua, right before RU was shut down due to coronacrisis. We went for 100mg scale per sample, derived from this tester. PA-1 DC cells were grown in matrigels and these harbor only half of the sample per given mass, so scale on these samples was doubled.

MATERIALS

• Nuclease free extraction buffer (20mM HEPES, pH7.4, 1% TritonX-100, 500mM NaCl, 1x protease inhibitor); RNAsin 1:250 in extraction buffer, 1:1000 in wash buffer.
• Scale: 100mg per sample with 20ul anti-ORF1p beads (200mg with 40ul of beads on PA-1 DC).

METHODS

All buffers made with RNAse-free H₂0.

Samples

1. N2102Ep
2. N2102Ep
3. N2102Ep
4. N2102Ep
5. NTERA2
6. NTERA2
7. NTERA2
8. NTERA2
9. PA-1 PC
10. PA-1 PC
11. PA-1 PC
12. PA-1 PC
13. PA-1 DC
14. PA-1 DC
15. PA-1 DC
16. PA-1 DC

Experiment was performed essentially as stated in RNA extraction after cell powder IP, with the following remarks:

• We took 10 µl of input for protein analysis, 35 µl for RNA input (double amounts again for DC). RNA sample was then supplemented with 100% TRIzol and snap frozen in LN2.
• We saved the FT for possible protein analysis later. No FT fraction was stored for RNA analysis.
• During the final wash step, 10% of the beads was taken and eluted in 10 µl LDS (equal for DC). This ‘E’ fraction for protein analysis was stored without DTT.
• The remaining 90% of the beads was eluted directly in TRIzol (also equal for DC), and RNA was subsequently extracted from this fraction (~6 µl per sample). For each sample 1 µl was aliquoted for QC with bioanalyzer (see results). RNA samples were passed to the Boeke lab at NYU Langone.
• Additionally, the organic phase of the phasemaker tubes was also stored at -20, for future MS analysis.

Overview of stored fractions (all x16)
Protein (in box -20) 
- Input (10 µl = 2.5%)
- FT 
- Elution in LDS, no DTT (10% of beads)
- Organic phase of phasemaker tubes for MS analysis after protein precipitation 

RNA (in -80 box)
- Input (35 µl = 7.5-10%), 1:1 in TRIzol. RNA not extracted yet. 
- Elution (90% of beads). RNA extracted and split into a 1 µl aliquot for bioanalyzer pico QC and remainder for RNA seq. 

RESULTS

RNA QC: Samples 1-11 and 12-16

DISCUSSION