RNA sequencing, RNA immunoprecipitation, and plasmid barcode sequencing

Lysis
1. Collect 50mL OD600 0.6 and flash-freeze. 
2. Wash cells 2X with cold 5mL TBS, first removing residual YPD after 1’ spin (3K rpm on Beckman Allegra 6) and also remove residual TBS after decanting and spinning 1’.
3. Resuspend cells with 50uL VRC and 500uL FA lysis buffer plus 100U/mL RNasin and 2X protease inhibitors (Calbio Set IV); make sure to add VRC at the same time as    buffer.
4. Transfer cells to 500uL 2X-washed zirconium/silica beads, washing with 1mL FA lysis buffer plus 40U/mL RNasin and 1X protease inhibitors. 
5. Vortex cells 5X on high (setting 10) for 30”on Vortex Genie 2 with TurboMix Cell Disrupting Accessory, putting lysate on ice water for 1’ between cycles. Vortexing cycles and beads used can be optimized for strain/organism. These beads lysed our yeast strains better than glass beads.
6. Remove 500uL of lysate into new tube and DNase digest for 15’at room temperature  with 12.5uL 1M MgCl2, 2.5uL 1M CaCl2, 3uL RNasin, 1X protease inhibitors (5uL)  and 12uL RQ1 DNase. I remove lysate from ice (after last 1’ice incubation - longer   could be better) and add reagents to get the lysate closer to room temperature. 
7. Spin 10’ at full speed on microcentrifuge, transfer supernatant to new tube, spin 5’more, and transfer supernatant to new tube.
 

=> Now have INPUT (have ~500uL) - clean 25uL for RNA input with RNeasy MinElute cleanup column

IP
1. Add 200uL lysate to 300uL dilution buffer (FA buffer without detergents) with 2X protease inhibitors (10uL) and transfer this 500uL diluted lysate to 25uL 3X-washed GFP-Trap beads (500uL dilution buffer, magnetize, repeat for a total of 3X).
2. Rotate end-over-end for 1hr at 4°C. Overnight will yield more recovery but 1hr resulted in less cleavage of Ssd1-GFP. 
3. Wash sample/beads (4X with 230, 110, 110, 110 dilution buffer) on Extractman
4. Last well on Extractman has 15uL 2X sample buffer (RNase-free, no dye, no beta-mercaptoethanol) + 1X protease inhibitors for eluting. Once beads are in this last well, I transfer beads in 15uL 2X sample buffer to 35uL 2X sample buffer (total of 50uL) + 1X protease inhibitors.
5. Incubate beads at 70°C for 10’. Seventy degrees appears to elute all of the GFP-tagged protein.
6. Magnetize beads and remove supernatant.

=> Now have IP - clean all (except 5uL for western) for RNA IP with RNeasy MinElute cleanup column. Make sure you add 150uL water to ~50uL IP sample to dilute out 2X sample buffer, otherwise Qiagen RLT buffer with sample buffer will result in major precipitation. There will be minor precipitation with 200uL (sample plus extra water) but addition of EtOH after RLT will remedy this.

FA lysis buffer

50 mM HEPES-NaOH (pH 7.5)
140 mM NaCl
1 mM EDTA
1% v/v Triton X-100
0.1% w/v sodium deoxycholate

Stable up to 1 year at room temperature.