Cloning target sequence in pBait plasmid
A. Purification and Digestion of pBait
1. Transfect pBait empty plasmid in a ccdB resistant E. coli strain, such as One Shot™ ccdB Survival™ 2 T1R Competent Cells (Invitrogen).
2. Miniprep pBait using QIAprep® Spin Miniprep Kit (Qiagen) or similar method.
3. Digest pBait with SapI (NEB) restriction enzyme according to supplier’s protocol.
4. Optional: gel purify the 4770 bp backbone using QIAquick® Gel Extraction Kit (Qiagen) or similar method.
B. Design and Preparation of Insert
PROBER use 3 concatemers of the target DNA or motif by default, but single copy, duplicate, and 4 or more concatemers can also be used with variable efficiency. PROBER signal will be fade for inserts size longer than than ~80 bp. Inserts <80 bp can be generated by annealing two complementary single stranded oligos, and adding complementary sticky ends to the ends. SapI digestion of pBait generates 5’GAG overhang within the upstream UAS site and 5’CGG overhang just ahead of the downstream UAS site, so add 5’CTCCG to the first oligo and 5’CCG/3’CG to the second complementary oligo to resore the UAS sites and generate stocky ends. This will place the insert immediately next to the nearest UAS sites on both ends. An example is provided below:
5’ CTCCG---------------------------- 3’
3’ GC----------------------------GCC 5’
C. Annealing oligos
1. Combine both oligos in the following 10 μl reaction: oligo 1 & 2 (20 uM each), 1X NEB restriction enzyme buffer (e.g., NEB CutSmart buffer), water.
2. Incubate the tube at 950C for 5 minutes in a thermal cycler or heatblock.
3. Slowly bring down temperature to RT by cooling down 0.1°C/sec in thermal cycler, or leave the tube at room temperature for 10 minutes.
4. Dilute the annealed oligos 50-fold with water.
5. Use 1-2 μl of the diluted annealed oligos directly in ligation reaction.
D. Ligation and transformation
1. Set up the following ligation reaction: SapI digested pBait (50-100 ng), annealed oligos (1-2 μl), 10X NEB T4 Ligase Buffer (2 μl), NEB T4 Ligase (1 μl), nuclease-free water (adjust to 20 μl final).
2. Incubate ligation reaction 10 minutes at RT, following transformation into E. coli DH5α or similar chemically competent cells and plating on LB plates containing 30 µg/mL kanamycin.
Cell culture and transfection
1. A night before transfection, plate HEK293T cells in 10 cm (for PROBER-WB) or 15 cm (for PROBER-MS) cell culture dishes with DMEM supplemented with 10% FBS and 1% penicillin–streptomycin (optional: plates can be coated with coating buffer containing Poly-L-lysine for better cell attachment).
2. Next day when cells are ~90% confluent, perform transfection (scale up 2X for 15 cm dishes).
a) Make transfection premix 1: 500 μl Opti-MEM, 32 μl P3000 enhancer, 10 ug pBait-YY1, 3 ug pSprayer, 3 ug pDriver, incubate 5 minutes in RT.
b) Make transfection premix 2: 500 μl Opti-MEM, 32 μl Lipofectamine L3000 reagent.
c) Combine transfection premixes 1 and 2, mix well.
d) Incubate at RT for 15-20 minutes.
e) Gently add 1 ml transfection mix to the plate containing cells, return plate to incubator.
3. Optional: Media chan be changed after 4-6 hours of transfection.
4. After 20 hours (at least 18 hours, at most 24 hours) of transfection, replace media with DMEM (with 10% FBS and 1% penicillin–streptomycin) containing 50 mM Biotin, return plates to incubator.
5. After 5 hours of adding biotin, remove plates from incubator.
6. Wash plates with 5 ml cold DPBS, aspirate DPBS.
7. Harvest cells with a cell scraper in 1 ml cold PBS and transfer cells to 1.7 ml centrifuge tube (cell dissociation reagents such as trypsin can be used without affecting PROBER results).
8. Spin down cells in 40C at 3000 rpm (~850 rcf), discard DPBS.
9. Proceed immediately with pulldown or freeze pellets in -800C for indefinite period.
Cell lysis (scale up 2X for 15 cm dishes)
1. Thaw cell pellets on ice if cells were frozen after harvesting.
2. Resuspend cells in 500 μl Lysis Buffer by pipetting up and down 10 times (note: the pellet may not resuspend fully).
3. Add 40 μl 25% Triton X-100, mix by brief vortexing.
4. Sonicate samples for 10-20 seconds at 10% amplitude in a Branson Digital Sonifier (or similar sonicator) using a 2.4 mm microtip, repeat post-cooling if lysate is not clear.
5. Spin lysate at maximum speed for 10 minutes at 4oC to remove cell debris.
6. Transfer clear lysate to a fresh tube avoiding the slimy pellet.
7. Add 500 μl-1 ml lysis buffer (or 50 mM Tris, pH-7.5, for less stringent condition) to dilute lysate.
8. Load lysate to 3K Omega™ Microsep® centrifugal devices (or similar tubes), spin at 4oC according to manufacturer’s protocol until the volume reduces to ~500 μl.
9. Pipette 2-3 times and collect the lysate from top chamber.
10. Optional: perform protein assay using Pierce™ Reducing Agent Compatible BCA Protein Assay Kit (or similar reducing agent compatible reagent), normalize concentration across samples using lysis buffer.
11. Save 10-25 μl lysate for analysis by western blot.
1. Prepare Streptavidin MyOne C1 dynabeads (50 μl beads for <2 mg lysate, 100 μl beads for 2 mg and above).
a. Aliquot appropriate volume in a 1.7 ml microcentrifuge tube.
b. Magnetize beads using a DynaMag-2 magnetic bead-separator (or similar instrument), aspirate the supernatant.
c. Wash with 50 mM Tris, pH-7.5: remove tube from magnet and resuspend beads in 1 ml buffer by piperring up and down 10 times. Magnetize and aspirate buffer.
d. Wash 1-2 times with 1 ml lysis buffer following the same method, remove buffer.
e. Resuspend beads in initial volume of lysis buffer (i.e., 50 μl lysis buffer for 50 μl beads).
2. Add pre-washed beads to lysates 1.5 ml in Eppendorf Protein LoBind tubes (50 μl beads for <2 mg lysate, 100 μl beads for 2 mg and above).
3. Rotate for 2 hours at room temperature or overnight at 40C in a Revolver™ Adjustable Rotator (The Lab Depot) or similar instrument.
1. Remove tubes from rotator and manetize beads, remove the lysate
(optional: save the lysate as “unbound fraction” for western blot).
2. Wash beads with Wash Buffer 1: resuspend beads in 1 ml buffer and mix by pipetting up and down. Rotate tubes in rotator for 10 minutes at room temperature. Magnetize and and aspirate supernatant.
3. Repeat washing with 1 ml Wash Buffer 1 (rotate 10 minutes).
4. Wash with 1 ml Wash Buffer 2 (rotate 10 minutes), remove supernatant.
5. Wash with 1 ml Wash Buffer 3 (rotate 10 minutes), remove supernatant.
6. Wash with 1 ml 50 mM Tris, pH-7.5 (rotate 10 minutes) by pipetting up and down for 10 times.
7. Optional: repeat Tris wash twice more times (specially for on-bead mass spectrometry sample processing).
8. Remove buffer and spin tubes briefly at low speed (< 5000 rpm) to collect all liquid at the bottom.
9. Magnetize and remove all residual buffer using a P20 (2-20 μl) pipette tip.
1. Add 50 μl freshly prepared elution buffer to each tube.
2. Place tubes in an eppendorf thermomixer (or similar instrument) and shake at 95oC at 1000 RPM for 15 minutes (place thermomixer lid or use tube caps to prevent tubes from popping).
3. Spin tubes briefly at low speed to collect all buffer at the bottom.
4. Magnetize tubes and transfer eluate to fresh tubes.
5. The eluate can be used for analysis immediately or stored at -20oC for indefinite period.
KinFisher FLEX Protocol (after lysis step)
1. Load 1 ml Wash Buffer 1 (2 plates), Wash Buffer 2, Wash Buffer 3, and 50 mM Tris, pH-7.5 in 96-well Kingfisher FLEX deep well plates.
2. Load appropriate volume of lysate from lysis step in a 96-well Kingfisher FLEX deep well plate.
3. In the same plate, add approipriate volume of pre-washed beads to the lysates.
4. Load comb, tip plate, and all deep well plates in Kingfisher FLEX instrument.
5. Perform binding and washes using the following program:
a) Binding: 2 hours (Bottom mix - 30 sec, Half mix - 1 min, Slow - 18 min 30 sec, repeat 6 cycles).
b) Wash with Wash Buffer 1: Release beads, Half mix - 2 min, Medium - 8 min.
c) Wash with Wash Buffer 2: Release beads, Half mix - 2 min, Medium - 8 min.
d) Wash with Wash Buffer 3: Release beads, Half mix - 2 min, Medium - 8 min.
e) Wash with 50 mM Tris, pH-7.5: Release beads, Half mix - 2 min, Medium - 8 min (leave beads in Tris buffer).
6. Collect beads from Tris plate and perform elution as mentioned before.
Quality check by WB
1. Run 10 μl eluate alongside saved lysates as input (5 μl of 1/5 diluted lysate) and a molecular weight marker in a NuPAGE™ 4-12% Bis-Tris gel.
2. Transfer to nitrocellulose (or PVDF) membrane and probe with appropriate primary antibodiy together with anti-YY1 and anti-HA antibodies.