Summary of a-ORF1p RNA extractions
Used cell lines:
• N2102Ep
• NTERA2
• PA-1 PC
• PA-1 DC
All in quadruplicates, 100mg scale. DC done with double amount because grown in Matrigel. Hence powder is approximately 100% diluted compared to other powders.
Extraction buffer: regular L1 extraction buffer with 1:250 RNasin and 1:1000 RNasin in wash buffer. 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 took saved the FT for possible protein analysis later. No FT fraction was stored for RNA analysis.
Anti-ORF1p IP was performed for 30’ with 20 µl beads per 100 mg (meaning 40 µl beads for DC). 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. 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)
o Input (10 µl = 2.5%)
o FT
o Elution in LDS, no DTT (10% of beads)
o Organic phase of phasemaker tubes for MS analysis after protein precipitation
• RNA (in -80 box)
o Input (35 µl = 7.5-10%), 1:1 in TRIzol. RNA not extracted yet.
o Elution (90% of beads). RNA extracted and split into a 1 µl aliquot for bioanalyzer pico QC and remainder for RNA seq.