TITLE: FlowFISH with PrimeFlow
AUTHORS: Ronghao Zhou, Jesse Engreitz

[DESCRIPTION]
Detect RNA expression in single cell with PrimeFlow

[STEPS]
SECTION: Before start:
1. Make sure the heat block temp is set to 40ºC using the digital monitor. The heat block should be ON at least a day in advance to stabilize temperature

SECTION: Before start:
2. Turn on Hyb oven (use to pre-warm target probe diluent)
SECTION: Before start:
3. Make FACS staining buffer: 0.5% BSA in PBS, filter sterilized
-       10% BSA stock: 3g BSA (light sensitive powder in 4ºC MISC) + 30mL PBS
-       Store at 4ºC
SECTION: Before start:
4. Set vortex to ~1500RPM 
SECTION: A.	Fixation and permeabilization
5. -       leave ~100µL when removing buffer, vortex to resuspend pellet
-       invert to mix when adding 1mL and tap the bottom if some cells stuck
-       pipette up and down to mix for fixation, permeabilization, and hybridization      
-       may be in bulk, use volumes that cells don’t exceed 10M cells/mL
-       pre-warm PrimeFlow RNA Wash Buffer to room temperature
SECTION: A.	Fixation and permeabilization
6. Aliquot 5-10M cells in PBS per sample
SECTION: A.	Fixation and permeabilization
7. Add 1mL PBS per sample, pipette to mix, and spin down at 500g at 4ºC for 5min, discard supernatant, resuspend in ~100µL by vortexing gently
SECTION: A.	Fixation and permeabilization
8. Prepare Fixation Buffer 1 by mixing equal parts of PrimeFlow RNA Fixation Buffer 1A and PrimeFlow RNA Fixation Buffer 1B
-       need 1mL per sample: 500µL Buffer 1A + 500µL Buffer 1B
-       mix gently by inverting, don’t vortex or vigorously shake, prepare fresh
-       keep at 4ºC
SECTION: A.	Fixation and permeabilization
9. Add 1mL Fixation Buffer 1, pipette to mix, incubate in dark (put cardboard box on it) rotating on rotator for 30min at 2–8°C in cold room
-       fixation time is critical, do NOT overshoot since the cells will lose integrity
SECTION: A.	Fixation and permeabilization
10. Prepare 1X RNA Permeabilization Buffer with RNase Inhibitors by diluting PrimeFlow RNA Permeabilization Buffer (10X) and RNase Inhibitors (100X) with RNase-free water
-       need 2mL per sample: 200µL Perm Buffer + 1.78mL H2O + 20µL RNase Inhibitor
-       mix gently by inverting, don’t vortex or vigorously shake, prepare fresh
-       keep at 4°C 
SECTION: A.	Fixation and permeabilization
11. Spin down cells at 800g at 4ºC for 5min, discard supernatant
-       speed is critical here: slower spin will lose a lot of cells
SECTION: A.	Fixation and permeabilization
12. Add 1mL RNA Permeabilization Buffer with RNase Inhibitors, pipette to mix, and spin down at 800g at 4ºC for 5min, discard supernatant
SECTION: A.	Fixation and permeabilization
13. Repeat wash with 1mL RNA Permeabilization Buffer
-       set centrifuge to room temp
SECTION: A.	Fixation and permeabilization
14. Prepare 1X RNA Fixation Buffer 2 by diluting PrimeFlow RNA Fixation Buffer 2 (8X) with PrimeFlow RNA Wash Buffer
-       need 1mL per sample: 125µL Fix Buffer 2 + 875µL RNA Wash Buffer
-       mix gently by inverting, don’t vortex or vigorously shake, prepare fresh
-       keep at room temp
SECTION: A.	Fixation and permeabilization
15. Add 1mL RNA Fixation Buffer 2, pipette to mix, and incubate for 60min in the dark at room temperature while rotating
SECTION: A.	Fixation and permeabilization
16. Spin down cells at 800g at room temp for 5min, discard supernatant, resuspend (~100µL) by vortexing gently
SECTION: A.	Fixation and permeabilization
17. Add 1mL PrimeFlow RNA Wash Buffer, invert to mix, and spin down at 800g at room temp for 5min, discard supernatant, resuspend (~100µL) by vortexing 
-       if in bulk, the cells should be transferred to the 1.5mL tubes from kit
SECTION: A.	Fixation and permeabilization
18. Repeat wash with 1mL RNA Wash Buffer
* can store samples overnight at 4ºC, then the last wash with RNase Inhibitors
SECTION: B.	Target Probe hybridization
19. Thaw Probe Sets (20X), including positive control (RPL13A, Type 4) on ice; 
pre-warm PrimeFlow RNA Target Probe Diluent to 40°C

SECTION: B.	Target Probe hybridization
20. take 2µL cells from “unstained” sample into 18µL PBS -> measure on countess with trypan blue and record cell amount and % live
-        critical that the residual volume after all washes be as close to 100μL as possible, use the markings on the 1.5mL tubes to assist
SECTION: B.	Target Probe hybridization
21. Dilute Probe Sets (20X) 1:20 in PrimeFlow RNA Target Probe Diluent, mix thoroughly by pipetting up and down
-       need 100µL per sample: 5µL Target Probe + 5µL RPL13A Probe + 90µL Diluent
-       for unstained sample: 100µL Diluent
-       keep at 40ºC
SECTION: B.	Target Probe hybridization
22. Add 100μL diluted Target Probe(s) into the cell suspension (~100µL), pipette to mix, briefly vortex, then incubate for 2h at 40°C
-       do not pipette solutions onto the walls of the tubes, and samples should be mixed well before incubating
-       vortex samples to mix every 30min
-       temp is critical for hybridization
SECTION: B.	Target Probe hybridization
23. Add 1mL PrimeFlow RNA Wash Buffer, invert to mix, and spin down at 800g for 5min, discard supernatant, resuspend (~100µL) by vortexing gently
SECTION: B.	Target Probe hybridization
24. 
Prepare PrimeFlow RNA Wash Buffer with RNase Inhibitors by diluting RNase Inhibitors (100X) with RNA Wash Buffer
-       need 1mL per sample: 10µL RNase Inhibitor + 990µL Wash Buffer
-       mix gently by inverting, prepare fresh
-       keep at room temp
SECTION: B.	Target Probe hybridization
25. Add 1mL PrimeFlow RNA Wash Buffer with RNase Inhibitors, invert to mix, and spin down at 800g for 5min, discard supernatant, resuspend (~100µL) by vortexing gently
-       Store samples overnight in the dark at 4ºC
SECTION: C.	Signal amplification
26. -       Pre-warm samples and PrimeFlow RNA Wash Buffer to room temperature
-       Pre-warm PrimeFlow RNA PreAmp Mix, PrimeFlow RNA Amp Mix, and PrimeFlow RNA Label Probe Diluent to 40°C
SECTION: C.	Signal amplification
27. Add 100μL PrimeFlow RNA PreAmp Mix into the cell suspension (~100µL), pipette to mix, briefly vortex, then incubate for 1.5h at 40°C
-       do not pipette solutions onto the walls of the tubes, and samples should be mixed well before incubating
SECTION: C.	Signal amplification
28. Add 1mL PrimeFlow RNA Wash Buffer, invert to mix, and spin down at 800g for 5min, discard supernatant, resuspend (~100µL) by vortexing gently
SECTION: C.	Signal amplification
29. Repeat wash two times with 1mL RNA Wash Buffer, for a total of three washes
SECTION: C.	Signal amplification
30. Add 100μL PrimeFlow RNA Amp Mix into the cell suspension (~100µL), pipette to mix, briefly vortex, then incubate for 1.5h at 40°C
-       do not pipette solutions onto the walls of the tubes, and samples should be mixed well before incubating
SECTION: C.	Signal amplification
31. Add 1mL PrimeFlow RNA Wash Buffer, invert to mix, and spin down at 800g for 5min, discard supernatant, resuspend (~100µL) by vortexing gently
SECTION: C.	Signal amplification
32. Repeat wash with 1mL RNA Wash Buffer
SECTION: C.	Signal amplification
33. Dilute PrimeFlow RNA Label Probes (100X) 1:100 in PrimeFlow RNA Label Probe Diluent
-       need 100µL per sample: 1µL Label Probes + 99µL Diluent
-       keep at 40ºC
SECTION: C.	Signal amplification
34. Add 100μL diluted Label Probes into the cell suspension (~100µL), pipette to mix, briefly vortex, then incubate for 1h at 40°C
-       do not pipette solutions onto the walls of the tubes, and samples should be mixed well before incubating
SECTION: C.	Signal amplification
35. Add 1mL PrimeFlow RNA Wash Buffer at room temp, invert to mix, and spin down at 800g for 5min, discard supernatant, resuspend (~100µL) by vortexing gently
SECTION: C.	Signal amplification
36. Repeat washes with warm (35ºC) 1mL RNA Wash Buffer 5 times
SECTION: C.	Signal amplification
37. Add 1mL FACS staining buffer (or PrimeFlow RNA storage buffer), invert to mix, and spin down at 800g for 5min, discard supernatant, resuspend (~100µL) by vortexing gently
-       Samples can be stored in the dark at 4ºC for up to three days before analysis
-       QC: take stained vs unstained samples and check fluorescence with microscope
SECTION: D.	Flow cytometric analysis
38. Add 100µL FACS buffer to cells, transfer all to filter, quick spin
SECTION: D.	Flow cytometric analysis
39. Add 100µL FACS buffer to cap to wash remaining cells from filter, quick spin
SECTION: D.	Flow cytometric analysis
40. FACS: 
-       400-500µL of an optimally concentrated sample (20M cells/mL) take ~30min
-       when low volume left, add ~200µL staining buffer can recover more cells
-       setup:
o   sort population should be <10% of unstained sample
o   exclusion should be <5% of stained sample
        §  if more than 5%, increase voltage or decrease compensation
o   compensate for each gene: AF647 over AF488
        §  RPL13A (AF488) expression = total RNA, compensate gene of interest expression (AF647) relative to total RNA
        §  average AF488 for Top & Bottom 25% AF647 should be within 10% of average AF488 for all sort population   

SECTION: E. gDNA Extraction after FlowFISH
41. -       Homemade ChIP Lysis Buffer:
o   1% SDS, 10mM EDTA, 50mM Tris-HCl, pH7.5
o   store at 4ºC
o   for 50mL
-       Make Lysis Buffer fresh or warm at room temp 30-60min to solubilize precipitate
-       Always include a no cell control until PCR to check contamination
-       Set thermocycler: 
o   65ºC Hold -> 65ºC 10min -> 37ºC Hold -> 37ºC 30min -> 65ºC Hold -> 65ºC 2h -> 95ºC 20min -> 4ºC Hold
-       Protocol for 1M cells
SECTION: E. gDNA Extraction after FlowFISH
42. Spin cells down for 10min at 800g at 4ºC, remove supernatant
SECTION: E. gDNA Extraction after FlowFISH
43. Resuspend cells in 70µL ChIP Lysis Buffer, transfer to 96-well plate
SECTION: E. gDNA Extraction after FlowFISH
44. Incubate at 65ºC for 10min
SECTION: E. gDNA Extraction after FlowFISH
45. When samples cool to 37ºC, add 2µL RNase Cocktail (Invitrogen AM2286) mix well by pipetting
SECTION: E. gDNA Extraction after FlowFISH
46. Incubate at 37ºC for 30min
SECTION: E. gDNA Extraction after FlowFISH
47. Add 10µL Proteinase K (Thermo 25530049), mix well by pipetting
SECTION: E. gDNA Extraction after FlowFISH
48.  Incubate at 65ºC for 2h, then 95ºC for 20min
SECTION: E. gDNA Extraction after FlowFISH
49. Store at 4ºC (can store overnight)
SECTION: E. gDNA Extraction after FlowFISH
50. Beads clean with 0.7X AMPure beads: 70µL AMPure beads for ~100µL samples
a.    warm AMPure beads to room temp
b.    add beads, pipette to mix, and bind DNA for 2min
c.     wash 3 times with 150µL 70% EtOH
d.    let beads dry for 5-10min after 3rd wash
e.    elute with ~40-50µL H2O, elute for 2min, transfer to a new plate/tube