N2102Ep, NTERA anti-ORF1 and anti-ORF2 IPs for PacBio sequencing
500mg scale: for each 500mg IP, do as 2x 250mg IPs and combine them at RNA purification step
Use 20ul beads per 100mg IP (100ul beads for 500mg)
Cell lines 1) N2102Ep (duplicates of anti-ORF1 and anti-ORF2 IPs)
2) NTERA (duplicates of anti-ORF1 IP only)
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors). Add RNasin to extraction buffer (1:250) and wash buffer (1:1000)
Anti-ORF1 and anti-ORF2 IPs
Weigh out 8x 250mg of N2102Ep and 4x 250mg of NTERA
Add 1000ul of extraction buffer with protease inhibitors and RNasin to each tube
Sonicate samples for 5x 2 sec at 2 Amp (each cycle, the energy output for 250mg samples: 14-16J); Repeat once; total energy 28-32J; put tube on ice between two rounds of sonication
Spin @ 21k rcf, 4°C for 10' (Eppendorf Centrifuge 5424R)
Take 12.5ul (~1%) of clarified whole cell lysate (input) from each replicate, snap freeze and store in -80°C freezer (may not need this fraction for anything)
Set up 250mg anti-ORF1 or anti-ORF2 IPs with 50ul of beads
• N2102Ep anti-ORF1: 4x 250mg
• N2102Ep anti-ORF2: 4x 250mg
• NTERA anti-ORF1: 4x 250mg
IP @ 4°C for 30'
Wash beads 3x 1ml with extraction buffer
Switch beads to fresh tubes at 2nd wash step
At the last wash, transfer 10% of the beads to a new tube, combine two replicates of each 500mg IP (5%x 500mg=25mg equivalent); use for anti-ORF1 Western
Elute in 250ul of Trizol: add Trizol and vortex for 1’
RNA extraction
all steps from here may be performed at RT if not mentioned otherwise
Vortex for 1min, place the tubes aside (can be on ice or RT, Trizol extract is stable at RT) Spin the phasemaker tube at 16k RCF, 30 sec
Add 50ul chloroform and 25ul nuclease free H2O to the phasemaker tube; Do this immediately before adding Trizol elutions
Collect the elution (pulse spin, place on magnet) and transfer to the phasemaker tube
Mix by hand, vigorously for 15 sec
Incubate 2 min @ RT with end-over-end mixing (this step is almost certainly dispensable for IP - it is supposed to be time for RNPs to dissociate.)
Spin 16k RCF, 4C (not important, Trizol extract is stable at RT), 5 min
Transfer the aqueous phase to a new tube by pressing pipette tip against wall of tube; avoid puncturing the Phasemaker gel
Pool two replicates of 250mg IP (same cell line, same beads) together here to produce one 500mg IP; check the total volume of combined aqueous phase
Check the volume (~170ul each, 340ul combined)
Add an equal volume of 100% EtOH to each sample and mix thoroughly, vortex and pulse spin
Check the volume again (340ul x 2+ 680ul)
Transfer the mixture to a Zymo-Spin IC column in a collection tube and centrifuge @ 16,000rcf for 30 seconds • The maximum capacity of each Zymo column is 700ul. To process sample >700ul, reload the sample and spin again
DNase I treatment
• Add 400ul RNA Wash Buffer to the column and centrifuge @ 16,000rcf for 30 sec • In an RNase-free tube, add 5ul DNase I 95U/ul), 35ul DNA Digestion Buffer and mix. Add the mix directly to the column • Incubate @ RT (20-30C) for 15 minutes
Add 400ul Direct-zol RNA Prewash to the column and centrifuge @ 16,000rcf for 30 seconds. Discard the FT and repeat this step. Use the vacuum-trap system to clear the liquid from the tubes both times
Add 700ul RNA Wash Buffer to the column and centrifuge for 2 minutes @ 16,000rcf to ensure complete removal of the wash buffer. Transfer the column carefully into an RNase-free tube.
Collect RNA samples
To elute RNA, add 6ul of RNase-Free Water directly to the column matrix and centrifuge @ 16,000rcf for 30 seconds.
Take 1ul aliquot for pico chip bioanalyzer analysis.
Store the two sets (1ul and 5ul for each) of tubes in -80°C freeze
1ul for pico chip bioanalyzer analysis; 5ul for PacBio sequencing
Send samples to Prescott Deininger @ Tulane Cancer Center
Anti-ORF1 Western
Elute beads with 10ul of 1.1x LDS
Add 0.5ul 1M DTT
Heat samples @ 70°C for 10'
Run a 4-12% Bis-Tris gel (25ug if not noted)
Wet transfer, 70V for 2h
Block the membrane in TBST/5% milk @ RT, 1hr
Anti-ORF1 Western (Abmart, 4H1, 2mg/ml)
• Primary Ab: Mouse anti-ORF1 antibody, 1:5,000, 4°C, overnight
• Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr