TITLE: single-cell TChIC for Zebrafish 
AUTHORS: Vivek Bhardwaj, Alberto Griffa, Helena Viñas Gaza, Peter Zeller, Alexander van Oudenaarden

[DESCRIPTION]
Establishing a cell-type-specific chromatin landscape is critical for the maintenance of cell identity during embryonic development. However, our knowledge of how this landscape is set during vertebrate embryogenesis has been limited, due to the lack of methods to jointly detect chromatin modifications and gene expression in the same cell. Here we present a multimodal measurement of full-length transcriptome and chromatin modifications in individual cells during early embryonic development in zebrafish using a novel protocol: single-cell transcriptome with chromatin immunoclevage (scTChIC)

[BEFORE_START]
Reagents, tips and tubes should be RNAse and DNAse-free, and ideally DNA-low-binding as well.

[STEPS]
SECTION: Embryo Dissociation
1. Obtain Zebrafish embryos. Grow them at 28.5 °C in a 10cm diameter petri dish in ~10 mL of fresh E3 media in an incubator. 

SECTION: Embryo Dissociation
3. Dechorionation
Transfer the embryos to a glass beaker with a plastic Pasteur pipette. Remove as much E3 as possible without exposing the embryos to air. Add ~5 mL of  2 micromolar (µM) Pronase Solution. Incubate at 28.5 °C for 3 min checking constantly for cracks in the chorion. If the chorions are still intact after the 3 minutes, incubate for longer (1 min - 2 min). At the first sign of the chorions cracking, proceed to the next step.

SECTION: Embryo Dissociation
4. Transfer the dechorionated embryos with a glass pipette to 1.5mL protein low-binding tubes. Use multiple tubes so that each tube only contains at most ~50 embryo (less at later timepoints).
SECTION: Embryo Dissociation
5. Wash the samples with 500 µL of Suspension Media at Room temperature
SECTION: Embryo Dissociation
1.3. Sort our the unfertilized embryos from the fertilized ones using a plastic Pasteur pipette. This can be done at any time between the collection of the eggs and their dissociation, but it's advisable to do it very early (~1hpf) because of the ease of distinguishing fertilized and unfertilized.
SECTION: Embryo Dissociation
1.1. If planning to incubate the embryos for a long time, check the water level periodically for evaporation. You might want to add a beaker with water just to provide a bit more moisture.
SECTION: Embryo Dissociation
1.2. Check on the embryos periodically. Count them and decide if and how to split them between one or multiple timepoints.
Optionally, take representative pictures of the embryos
SECTION: Embryo Dissociation
3.1. Dilute the Pronase with serial dilutions with a large (enough to fill 90% the beaker) volume of E3 media. Pour away as much liquid as possible without leaving the embryos exposed to air, and repeat this washing step another 3-4 times. Swirl the beaker to release the embryos from their chorions.
SECTION: Embryo Dissociation
4.1. Deyolking
Remove as much E3 as possible (without leaving the embryos exposed to air). Add 200 µL of Calcium-free Ringers solution.

SECTION: Embryo Dissociation
4.2. Gently pipette up and down with a P200 pipette to deyolk (~15 times).

 

SECTION: Embryo Dissociation
4.3. Let sit in Ringer’s solution for approximately 5 min at Room temperature.  
SECTION: Embryo Dissociation
4.4. Centrifuge the samples at 600 rcf, 3 min with a Swinging Buckets Centrifuge
 

SECTION: Embryo Dissociation
2. 1 hour before the start of dissociation prepare and/or thaw the reagents required:

1. fresh E3 media (1 L or more)

2. Pronase solution (1mg/mL, dissolved in E3 media)
  

3. Calcium-free Ringer’s solution (prepared following ZFIN recipe book, 5th Edition https://zfin.atlassian.net/wiki/spaces/prot/pages/362220041/RINGER+S+SOLUTIONS)

4. FACSmax (used for timepoints lower than 10hpf)
 
 

5. Protease solution (used for timepoints from 10hpf (included) onwards)
25mL 0.5% Trypsin-EDTA
5mL 10X PBS0
20mL sterile water

6. Protease Stop solution (used for timepoints from 10hpf (included) onwards)
3ml FBS
30μL of 2M CaCl2
5.9mL sterile water
1mL 10X PBS0 (PBS0 is PBS without Ca+ and Mg+)

7. Suspension media (1mL FBS + 9ml PBS0). PBS0 is PBS without Ca+ and Mg+

Cell Tracer dyes and rat serun to stop staining (depending on whether multiple timepoints will be mixed in a single tube or not, see flowcharts below):       
 

 
 


After the dissociation is complete, there are a few possible routes that can be taken (requiring tweaks to the solutions). The flowchart below illustrates the 2 main choices and their required changes: 
 

The choice of protocol will determine which solutions to prepare and whether to also procure the cell tracer dyes. Below is a more detailed overview of which solutions are needed and for which steps according to your choices. We used the two approaches in the bottom row in our datasets. 
 




How to prepare the various Washing Buffers (CellTrace Staining included)
If you are not planning on using the Celltrace dyes to combine multiple samples, see alternative option below at step 2.1

Prepare Wash Buffer(w/out Spermidine), regular Wash Buffer, Wash Buffer1 (w/out Spermidine) and regular Wash Buffer 1 (WB1). Not all of these need to prepared now, but for the sake of completeness, they are all presented here. 

We suggest preparing the Wash buffers in the following order: prepare 50mL of Wash Buffer(w/out Spermidine) and take 10mL aside to make WB1 (w/out Spermidine). Use the remaining 40mL to make regular WB, and from regular WB, prepare WB1 as needed. 

Depending on whether the objective is to capture cells or nuclei, switch out the Tween-20 for 10% Saponin.
(Dissolve fresh 10% Saponin in PBS0. Store at 4 °C , use within 2 days. )

For all Wash Buffers: prepare on ice, store at 4 °C, use for up to 2 days

Wash Buffer(w/out Spermidine) 
       
  Wash Buffer MINUS 
  Volume
  50 mL
  
  H2O
  RNAse free water
  47.8
  mL
  
  HEPES pH 7.5 (1M)
  1
  mL
  
  NaCl(5M)
  1.5
  mL
  
  Saponin 10%
  250
  µl
  
  Protease inhibitor
  1
  complete tablet
    
  


from the Wash Buffer(w/out Spermidine), prepare the Wash Buffer(w/out Spermidine) 1. We suggest preparing it on a per-needed basis to save on the RNAse inhibitor.  
       
  Wash Buffer MINUS 1
  Volume 1 mL
  Volume 1.5mL
  Volume 7,5
  
  Wash Buffer
  MINUS
  1mL
  1.5 mL
  7.5 mL
  
  EDTA 0.5M
  4 µl
  6 µl
  30 µl
  
  RNAseINH
  25 µl
  37.5 µl
  187,5 µl
    
  


From the remaining Wash Buffer(w/out Spermidine), prepare 40mL of the regular Wash Buffer (by adding the Spermidine).
       
  regular
  WB
  Volume 40mL
  
  Wash Buffer
  MINUS
  40mL
  
  Spermidine solution 
  2.88 µl
    
  

From the regular Wash Buffer, prepare the Wash Buffer 1. We suggest preparing it on a per-needed basis to save on the RNAse inhibitor.  

       
   
  WB1
  
  regular WB
  2.5 mL
  
  EDTA (0.5M)
  10 µl
  
  RNAse inhibitor (1:40)
  62,5 µl
    
  

SECTION: Embryo Dissociation
6. Dissociation: 
Resuspend cells in 200 µL of either FACSmax (for timepoints up to 8hpf (included)), or Protease Solution (for timepoints later than 10hpf (included). Incubate on shaker at  
Resuspend up and down gently every 2min.

Check under the microscope that the suspension is single cell! If not keep longer. 
 

If the solution is already single cell, interrupt the shaking and proceed to the next step. 
SECTION: Embryo Dissociation
7. Collect and filter the single cell solution into a 1.5 mL prot-low binding tube using the blue cap from FACS tubes (35 um) and centrifuge at 600 rcf, 3 min with the swinging buckets centrifuge. 
SECTION: Embryo Dissociation
8. Resuspend the cell pellet in 500 µL of Suspension media at Room temperature . Take a10 µL aliquot to count the number of cells. Centrifuge the rest at 600 rcf, 3 min with the swinging buckets centrifuge. 

SECTION: Embryo Dissociation
9. Resuspend the pellet in 500 µL of WB1 Full (remember the 1:40  ). 
Keep on on ice until all the timepoints have been collected.

SECTION: DAY 1: Cell Staining and Antibody Incubation
10. Centrifuge all samples at 600 rcf, 3 min with the swinging buckets centrifuge. 
Wash each sample with 200 µL of WB1 (w/out Spermidine) (with 1:200  ), and centrifuge them again at 600 rcf, 3 min with the swinging buckets centrifuge. 

Skip till Step 13 if you are not planning on staining your samples!

SECTION: DAY 1: Cell Staining and Antibody Incubation
11. Resuspend each sample in 1 mL of WB1 (w/out Spermidine). Add to each sample 1 µL of one or a combination of the CellTrace Dyes:  
     

We suggest consistently using the same staining setup between replicate experiments to speed up the configuration of the FACS Sorting Layout.
SECTION: DAY 1: Cell Staining and Antibody Incubation
12. Add 70 µL of Rat Serum for each 1 mL of the staining volume (7% of staining volume) to each sample and incubate at Room temperature for 5 min to stop the staining. 
SECTION: DAY 1: Cell Staining and Antibody Incubation
11.1. Vortex each sample well!
SECTION: DAY 1: Cell Staining and Antibody Incubation
11.2. Incubate the samples in the dark on ice for 20 min

SECTION: DAY 1: Cell Staining and Antibody Incubation
11.3. In the meantime, prepare/thaw the   (which will stop the staining)

SECTION: DAY 1: Cell Staining and Antibody Incubation
12.1. Centrifuge the samples at 600 rcf, 3 min, 4 °C with the swinging buckets centrifuge. 
SECTION: DAY 1: Cell Staining and Antibody Incubation
13. Wash each sample with 200 µL of WB1 Full (with 1:200  ) , and centrifuge the samples at 600 rcf, 3 min, 4 °C with the swinging buckets centrifuge. 

SECTION: DAY 1: Cell Staining and Antibody Incubation
14. Resuspend and collect the samples in 1 tube with WB1 Full (with 1:40  ). Calculate the final value so that the concentration of cells is approximately 1 Mil cells/mL, and the mixing ratios corresponds to the desired experimental mix. 
If planning to measure more than one histone modification in the same experiment, split the sample in the corresponding number of tubes. 

If using colors to mix samples, an unstained populations of carrier cells can be added to increase the cell number.
SECTION: DAY 1: Cell Staining and Antibody Incubation
15. Add to each sample the appropriate primary antibody, at the appropriate dilution. Incubate 5 min on a roller in the dark and at 4 °C. 

 


SECTION: DAY 2: Sort, Protein A-MN treatment and activation
17. Prepare 384-wells Plates
Before starting, make sure to have enough plates to sort into. These should be 384-wells hardshell plates (Biorad) containing at least 5uL of Mineral Oil
   and 0.05 µL of  WB2 containing 28ng/uL of Celseq2 adapters. 
 

SECTION: DAY 2: Sort, Protein A-MN treatment and activation
17.1. You can prepare the plates by first filtering the mineral oil with a  and then dispensing several (between 5 and 10) uLs of the filtered mineral oil in each plate.

 

SECTION: DAY 2: Sort, Protein A-MN treatment and activation
17.2. Next you should dispense in each plate, 0.05 µL of CelSeq2 adapters, diluted in WB2, in each well. We use a Mosquito Liquid Handling robot (SPT Labtech) to transfer 0.05 µL from each well of a master plate(containing the primers in WB2) to each well of the target plate(s) (up to 4 at the same time). To minimise evaporation, the Mosquito should be humidified to a humidify of ~ 
Keep the plates on ice if you plan to use them the same day, otherwise store them at -20 °C . When needed, thaw them ~ 30 min before the sort  on ice or on thermocyclers cooled to 4 °C.

WB2 
Wash Buffer Concentration Volume 50 mLVolume 10 mLH2O RNAse free water 47.8 mL9.56 mLHEPES pH 7.51M1 mL200 mlNaCl5M1.5 mL300 mlSpermidine solution  3.6 µl0.72 mlTween (10%)0.05%250 µl50 mlProtease inhibitor 1 complete tablet  

The CelSeq2 adapters were ordered from IDT and are distributed according to this layout (same as CelSeq2 and VASA-seq)    
SECTION: DAY 2: Sort, Protein A-MN treatment and activation
19. Resuspend the sample in 500 µL of WB (with 1:40  ) + ProteinA-MN (600uL/mL, add 5uL every 1mL of WB) and Hoechst dye.

 

Mix for 60 min at 4 °C on a roller in the dark.
SECTION: DAY 2: Sort, Protein A-MN treatment and activation
18. Centrifuge the antibody stained sample at 600 rcf, 3 min, 4 °C with the swinging buckets centrifuge. Respond the sample in 500 µL of WB (with 1:200  ). 

Centrifuge the sample again at 600 rcf, 3 min, 4 °C with the swinging buckets centrifuge.

 

20. Centrifuge the sample at 600 rcf, 3 min, 4 °C with the swinging buckets centrifuge. Resuspend the sample in 500uL of WB (with 1:200  ). 
20.1. Repeat this wash again.   

21. Filter the sample through the blue caps filter of FACS tubes. Proceed to sorting. Keep samples in the dark and on ice
22. Sort single cells/nuclei in 384 well-plates containing mineral oil and Celseq2 adapters in WB2. Use the Hoechst and Celltracer dyes staining to identify the cells and distinguish each timepoints. 
Label the plates as you sort them (We suggest adding Histone mark, Experiment, Sorting Order information in the labelling).
 

22.1. Keep plates on ice
Immediately after sorting, seal the plates with    
Once sealed, immediately centrifuge 2000 rcf, 2 min, 4 °C  the plate, then move it on coolblocks on ice, or on 384-wells thermocyclers set at 4 °C.

24. Dispense 0.1 µL of WB2 +  3 millimolar (mM) CaCl2 in each well to activate the MN.
We use a Nanodrop Liquid Handling machine for this step, and dispense into 2 plates, held in cool blocks, at the time.
As soon as plates have received the MNAse, seal each plate with Aluminium plate sealer and proceed to centrifuge them at 2000 rcf, 2 min, 4 °C . 
Transfer the plates to 384-wells thermocyclers set at 4 °C for a 30 min digestion.

 

 

25. Stop the digestion of the plates by adding 0.1 µL of MNase Stop Solution to each well.
(Remember to add the Thermolabile Prot K, if not done previously)

We use a Nanodrop Liquid Handling machine for this step, and dispense into 2 plates, held in cool blocks, at the time.

Once the dispension is complete, seal each plate with Alluminium plate sealer and proceed to centrifuge them at 2000 rcf, 2 min, 4 °C .
Once the centrifugation is complete, move plate to a 384-well thermocycler.

 

SECTION: DAY 2: Sort, Protein A-MN treatment and activation
16. 
 

25.1. Incubate the plate at 4 °C  for 20 min, then 37 °C for 120 min, then 55 °C for 20 min and then hold at 4 °C.
23. Prepare the WB2 + 3 millimolar (mM) CaCl2 and the Stop Solution to start and (then) stop the MN digestion. 

WB2 + CaCl2:
Prepare WB2 
Wash Buffer Concentration Volume 50 mLVolume 10 mLH2O RNAse free water 47.8 mL9.56 mLHEPES pH 7.51M1 mL200 mlNaCl5M1.5 mL300 mlSpermidine solution  3.6 µl0.72 mlTween (10%)0.05%250 µl50 mlProtease inhibitor 1 complete tablet  
Dilute CaCl2 solution ~1:1000 (or more) in the WB2 solution, to a final concentration of 3mM. 


MNase Stop Solution:
Stop SolutionConcentrationVolume (1 plate)Nº of platesH2O RNAse free water 50.25 µl EGTA0.5 M6 µl NP4010%11.25 µl Prot K TL 20 mg/mL7.5 µl MgCl21M0.94 µl 
 
  

 

26. Once done, freeze all plates in a -80 °C . The plates can then be thawed and processed at any time.
SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
27. Thaw the plates you plan to process on ice or at 4 °C on pre-cooled thermocyclers.
 

SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
28. Fragment RNA
Incubate the plates in a pre-warmed thermocycler at 85 °C for 2 min. 
Immediately move plates to a pre-cooled thermocycler at 4 °C. 
 

SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
29. Blunt End RNA fragments and Poly-A tailing
Prepare Mix1, and aliquot it in a 8-well strip tube to dispense with the Nanodrop. Keep on ice
   Standard amountsmix19T4 PNK1,210,8polyA polymerase 0,1751,575ATP (0.1mM)(1 in 100)0,887,875Tris pH82,825,2PEG80000,756,75BSA (200x)0,0750,675RNAse out19KCl 1M327DTT 0,1M218MgCl2 1M0,151,35H2O3,1228,08Per well in strip17,038125
  
  
 
     
 
  
SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
29.1. Dispense Mix1 with Nanodrop Liquid Handling Robot. Dispense 0.150 µL of Mix1 per each well. 
Once the dispension is complete, seal each plate with Alluminium plate sealer and proceed to centrifuge them at 2000 rcf, 2 min, 4 °C .
SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
29.2. Once the centrifugation is complete, move plate to a 384-well thermocycler. Incubate the plate at 37 °C  for 60 min, and then hold at 4 °C.
SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
30. First Strand cDNA synthesis of RNA fragments
 Prepare Mix2, and aliquot it in a 8-well strip tube to dispense with the Nanodrop. Keep on ice
   mix214SSIII19KCl 1M0,7510,5DTT 0.1M0,57dNTP0,7510,5PEG80000,57BSA (200x)0,050,7H206,4590,3Per well in strip16,875
  
  
  
SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
30.1. Dispense Mix2 with Nanodrop Liquid Handling Robot. Dispense 0.100 µL of Mix2 per each well.
Once the dispension is complete, seal each plate with Alluminium plate sealer and proceed to centrifuge them at 2000 rcf, 2 min, 4 °C .
SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
30.2. Once the centrifugation is complete, move plate to a 384-well thermocycler. Incubate the plate at 50 °C  for 60 min, and then hold at 4 °C.
SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
31. DNA fragment Blunt Ending
Prepare Mix3,  and aliquot it in a 8-well strip tube to dispense with the Nanodrop. Keep on ice
  mix39Klenow large0,3252,925NaCl 5M0,21,8T4 PNK0,3252,925TL Exo10,54,5dNTPs (10mM)0,3252,925Tris-HCl 7,5pH1,7515,75DTT 0,1M0,756,75ATP 100mM0,655,85MgCl2 1M0,252,25PEG80000,756,75BSA (200x)0,0750,675H209,181,9Per well in strip16,875
 
  
  
  
  
  
    
  
  
  
SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
31.1. Dispense Mix3 with Nanodrop Liquid Handling Robot. Dispense 0.150 µL of Mix3 per each well.
Once the dispension is complete, seal each plate with Alluminium plate sealer and proceed to  centrifuge them at 2000 rcf, 2 min, 4 °C .

SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
31.2. Once the centrifugation is complete, move plate to a 384-well thermocycler. Incubate the plate at 37 °C  for 30 min, then 75 °C for 20 min, and then hold at 4 °C.

SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
32. A-tailing DNA fragments
Prepare Mix4,  and aliquot it in a 8-well strip tube to dispense with the Nanodrop. Keep on ice
 
  mix49Taq 3600,21,8dATP0,21,8KCl 1M19,0PEG8000,756,8BSA (200x)0,0750,7H2O12,775115,0Per well in strip16,9
 
  
  
  
  
  

SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
32.1. Dispense Mix4 with Nanodrop Liquid Handling Robot. Dispense 0.150 µL of Mix4 per each well. 
Once the dispension is complete, seal each plate with Alluminium plate sealer and proceed to  centrifuge them at 2000 rcf, 2 min, 4 °C .

SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
32.2. Once the centrifugation is complete, move plate to a 384-well thermocycler. Incubate the plate at 72 °C  for 15 min, and then hold at 4 °C.

SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
32.3. Start thawing the adaptor master plate (on ice). It's advisable to centrifuge it at 2000 rcf, 2 min, 4 °C once it's thawed.
Start humidifying the Mosquito Liquid Handling robot (SPT Labtech).
SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
33. Adaptor Ligation DNA fragments
Using a Mosquito Liquid Handling robot (SPT Labtech) transfer 50nL of 5uM paired forked adaptors from each well of the adaptor master plate to the plate(s) being processed. 
 

These adaptors were purchased from IDT, paired (top and bottom strand) and annealed, diluted to the working concentrations and stored at -20C till used. Sequences and layout are in this table:  
A more complete explanation of their design is in the Supplementary Information of the sortChIC paper from where they are derived: 
Single-cell sortChIC identifies hierarchical chromatin dynamics during hematopoiesis
SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
33.1. Once the dispension is complete, seal each plate with Alluminium plate sealer and proceed to centrifuge them at 2000 rcf, 2 min, 4 °C .
SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
33.4. Dispense Mix5 with Nanodrop Liquid Handling Robot. Dispense 0.150 µL of Mix5 per each well.
Once the dispension is complete, seal each plate with Alluminium plate sealer and proceed to  centrifuge them at 2000 rcf, 2 min, 4 °C .

SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
33.5. Once the centrifugation is complete, move plate to a 384-well thermocycler. Incubate the plate at 4 °C  for 20 min, then at 16 °C for 960 min, then 65 °C for 10 min, and then hold at 4 °C.

SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
33.2.    Repeat the dispension with the other plates.
 

SECTION: DAY 3: RNA and DNA Fragment processing and DNA adapter ligation
33.3. Prepare Mix5, and aliquot it in a 8-well strip tube to dispense with the Nanodrop. Keep on ice
 
  mix59T4 ligase (40000U)3,531,5Adapter 5uM00MgCl2 1M0,353,15DTT 0,1M6,7560,75ATP 100mM0,353,15Tris pH7.5 1M0,454,05PEG80000,756,75BSA (200x)0,0750,675H202,8525,65Per well in strip16,959375
 
  
  
  
  
  
  
  

SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
34. Pooling:
Add 1uL of nuclease-free Water in each well of each plate. 

 

SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
35. Prepare Pooling Plate(s)
Label each pooling plates so that they can be identified and connected with the plate they are to collect. 
Pre-wet each pooling plate with 1 mL or 2 mL of filtered Mineral Oil. Tilt the plates until the oil has spread over the entire collection surface. We use  , but a clean, upside-down pipette tips box lid works as well .

SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
36. For each plate, pool the aqueous phase in 2mL DNA-low-binding tubes. Centrifuge the tubes at 2000 rcf, 1 min (this is just to separate the acqueous phase form the oil).

SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
37. Take the lower aqueous phase and move it into a 1.5 mL DNA-low binding tube.
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
38. Clean up DNA using DNA beads
Add 0.8X µl (depending on volume of each sample) of DNA beads diluted 1 in 8 with bead binding buffer (1 M NaCl, 20% PEG8000, 20mM Tris pH8, 1 mM EDTA) and mix by pipetting.
Take the beads out of the fridge ~ 30 min before using them
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
39. Resuspend in 17 µL of nuclease-free water (away from the magnet). 
Let elute for 2 min, then move back onto the magnet. Once the beads have coalesced on the side of the tube and the eluant is clear, move the eluant to a new tube.
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
40. Second Strand Synthesis:
on ice!
Add 2 µL of 10X SSS buffer. Mix by vortexing and spin down.
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
41. Add 1.3X µl of undiluted DNA beads (26 µL) and incubate for 15 min at Room temperature
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
34.1. Once the dispension is complete, seal each plate with Alluminium plate sealer and proceed to centrifuge them at 2000 rcf, 2 min, 4 °C .
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
35.1. Place each plate upside down over a pooling plate.
Centrifuge them at up to  2000 rcf, 2 min, 4 °C . Do not stack pooling plates on top of each other in the centrifuge. 

 

SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
37.1. Centrifuge the tubes 2000 rcf, 1 min again.
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
37.2. Take the lower aqueous phase and move it into a 1.5 mL DNA-low binding tube. During this pipetting step measure the volume.
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
38.1. Incubate for 15 min at Room temperature.
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
38.2. Move eppendorfs on a magnet rack and, once the beads have coalesced on the side of the tube and the supernatant is clear, remove the supernatant. 
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
38.3. Add 1 mL of 80% EtOH to each sample. Incubate for 30 s

SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
38.4. Remove the supernatant. 
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
38.5. Repeat the 80% EtOH wash   
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
38.6. Dry up the beads (until they should lose their shimmer, up to  10 min. Drying time is influenced by the humidity. Over and under drying leads to material loss!!)
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
40.1. Add 1 µL of SSS Enzyme and mix by gently flicking the tube. Spin down
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
40.2. Incubate at 
16 °C for 150 min, 65 °C for 20 min, then hold at 4 °C
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
41.2. Move eppendorfs on a magnet rack and, once the beads have coalesced on the side of the tube and the supernatant is clear, remove the supernatant. 
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
41.3. Add  1 mL of 80% EtOH to each sample. Incubate for 30 s

SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
41.4. Remove the supernatant. 
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
41.5. Repeat the 80% EtOH wash   
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
41.6. Dry up the beads (until they should lose their shimmer, up to  10 min. Drying time is influenced by the humidity. Over and under drying leads to material loss!!)
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
42. IVT
Prepare the IVT mix (thaw the reagents (except the T7 enzyme) ~30 min in advance):
 IVT Mix1 tubeN + 1 tubeATP2GTP2CTP2UTP2T7 Buffer2T7 Enzyme2
 
Add 12 µl of IVT mix to each tube of eluate with beads. 
Incubate on a thermocycler at 37 °C for 780 min, with lid at 70 °C. Then, hold at 4 °C

SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
41.7. Resuspend in 8 µL of nuclease-free water (away from the magnet). Let elute for ~ 2 min
SECTION: DAY 4: Pooling, Second Strand Synthesis, Amplification
41.1. Take out of the freezer the IVT reagents (except the enzyme) so they start thawing ~30min in advance of when they will be used.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
43. IVT bead cleanup
Move samples onto magnet rack. 
Move supernatant to a new tube, and add 1.3X Ampure RNA beads (26 µL) and incubate for 10 min at Room temperature
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
43.2. Add 1 mL of 80% EtOH to each sample. Incubate for 30 s

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
43.3. Remove the supernatant. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
43.4. Repeat the 80% EtOH wash   
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
43.5. Dry up the beads (until they should lose their shimmer, up to  10 min. Drying time is influenced by the humidity. Over and under drying leads to material loss!!)
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
43.6. Resuspend in 10 µL of nuclease-free water (away from the magnet). Let elute for ~ 2 min
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
43.1. Move eppendorfs on a magnet rack and, once the beads have coalesced on the side of the tube and the supernatant is clear, remove the supernatant. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
43.7. Place sample in magnet rack, once the beads have coalesced on the side of the tube and the eluant is clear, move the supernatant to a new tube.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
44. rRNA depletion
Transfer 6 µL of the aRNA to a new tube on ice, and freeze the remaining eluant at -80 °C

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
45. Prepare the Hybridisation buffer - oligos mix:
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
45.1. Prepare the Hybridisation Buffer. 
 

 

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
45.2. Mix the Hybridisation Buffer with the rRNA depletion Oligos (on ice)
 1 tuben+1 tubesHybridization Buffer2 uLrRNA-depletion oligos2 uL

The rRNA depletion oligos that we used for the Zebrafish Ribosomes are these (ordered from IDT and pooled together):   
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
45.3. Add 4 µL of the Hybridisation Buffer + rRNA depletion Oligos mix to each tube.
Spin down and cool °C.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
45.4. Incubate tubes on a thermocycler at 95 °C for 2 min, then decrease the temperature to 45 °C at a rate of 0.1C/s, then hold at 45 °C
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
46. Prepare the RNAse mix:
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
46.2.  Prepare the RNAse Mix.
 1 tuben+1 tubesEpicentre (Lucigen) RNaseH (Thermostable)2 uLRNAse buffer8 uL
 
  

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
46.1. Prepare the RNAse Buffer. 
 

 

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
46.3. Incubate the RNAse Mix at 45 °C for a few minutes (we suggest placing it in the same thermocycler as samples, once they are holding at 45C)

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
46.4. While keeping both samples and Hybridise Mix on the thermocycler at 45 °C, add 10 µL of the RNAse mix to each sample.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
46.5. Incubate the samples at 45 °C for 30 min.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
46.6. Spin down and cool on ice.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
47. DNAse treatment (removal of depletion oligos)
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
47.1. Prepare the DNAse Mix:
 1 tuben+1 tubesNuclease-free Water21 uLCaCl2 (10mM)5 uLPromega DNAse4 uL
   

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
47.2. Add 30 µL of DNAse Mix to each tube. Incubate at 37 °C for 30 min, then hold at 4 °C. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
47.3. Start thawing Ampure RNA beads at Room temperature  ~30 min before the Bead clean up.

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
48. Bead Cleanup
Add 80 µL of undiluted, pre-warmed RNA beads to each sample and incubate at Room temperature for 10 min.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
48.1. Move eppendorfs on a magnet rack and, once the beads have coalesced on the side of the tube and the supernatant is clear, remove the supernatant. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
48.2. Add  1 mL of 80% EtOH to each sample. Incubate for 30 s

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
48.3. Remove the supernatant. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
48.4. Repeat the 80% EtOH wash   
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
48.5. Dry up the beads (until they should lose their shimmer, up to  10 min. Drying time is influenced by the humidity. Over and under drying leads to material loss!!)
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
48.6. Resuspend in 6 µL of nuclease-free water (away from magnet). Let elute for ~ 2 min
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
48.7. Place sample in magnet rack, once the beads have coalesced on the side of the tube and the eluant is clear, transfer 5 µL of eluant to a new tube (on ice).
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
49. Adapter ligation
Pre-heat a thermocycler to 70 °C (with heated lid at 105 °C).
Add to each sample 1 µL of RA3(20 micromolar (µM)).

RA3 (ordered from IDT): /5rApp/TGGAATTCTCGGGTGCCAAGG/3SpC3/
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
49.1. Heat adapter-sample-mix at 70 °C for 2 min and then directly put on ice

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
49.2. Pre-heat a thermocycler to 25 °C  (with lid heated at 25 °C or without the lid closed).
Prepare the Ligation Mix (on ice):
 1 tuben+1 tubesNEB 10x T4 RNA Ligase Reaction Buffer1.1 µl NEB T4 RNA Ligase 2, truncated1.1 µl Invitrogen RNaseOUT1.1 µl H2O1.1 µl 
  
  

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
49.3.  Add 4 µL of the Ligation mix to each tube, mix well by pipetting and spin down (keep on ice)

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
49.4. Incubate on thermocycler at 25 °C for  60 min (lid heated at 25°C or without the lid closed), then hold at 4 °C .
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
50. cDNA synthesis
Pre-heat a thermocycler to 65 °C (with heated lid at 105 °C).
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
50.1. Prepare the dNTPs Mix (on ice):
  1 tuben+1 tubesPromega dNTP Mix (10 mM each)1 µl RTP (20 µM)2 µl 
  
  

RTP (ordered from IDT): GCCTTGGCACCCGAGAATTCCA
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
50.2. Add 3 µL of the dNTPs to each tube. 
Incubate on thermocycler at 65 °C for 5 min, then transfer tubes directly on ice.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
50.3. Prepare the Reverse Transcription Mix (on ice):
  1 tuben+1 tubesInvitrogen 5x FS Buffer 4 µl H2O1 µl Invitrogen 0.1M DTT1 µl Invitrogen RNaseOUT (40U/ µl)1 µl Invitrogen SS III (200U/ µl)1 µl 
 
    
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
50.4. Add 8 µL of the Reverse Transcription mix to each tube, mix well by pipetting and spin down (keep on ice).
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
50.5. Incubate in a thermocycler at 50 °C for 60 min , then 70 °C for 15 min, and then hold at 4 °C (with lid heated at 85 °C).
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
51. RNA degradation 
Add 1 µL of     to each tube.
Incubate on thermocycler at 37 °C for 30 min, then hold at 4 °C (with heated lid at 70 °C)
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
52. Bead Cleanup
Add 22 µL of undiluted, pre-warmed Ampure XP beads to each sample and incubate at Room temperature for 10 min.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
52.1. Move eppendorfs on a magnet rack and, once the beads have coalesced on the side of the tube and the supernatant is clear, remove the supernatant. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
52.2. Add 1 mL of 80% EtOH to each sample. incubate for 30 s

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
52.3. Remove the supernatant. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
52.4. Repeat the 80% EtOH wash   
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
52.5. Dry up the beads (until they should lose their shimmer, up to  10 min. Drying time is influenced by the humidity. Over and under drying leads to material loss!!)
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
52.6. Resuspend in 20 µL of nuclease-free water (away from magnet). Let elute for ~ 2 min
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
52.7. Place sample in magnet rack, once the beads have coalesced on the side of the tube and the eluant is clear, transfer 20 µL of eluant to a new tube (on ice).
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
51.1. Start thawing Ampure DNA beads at Room temperature  ~30 min before the next Bead clean up.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
52.8. Split the eluant in 2 tubes. Store half (10 µL) at -20 °C, and continue with the other 10 µL. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
53. PCR amplification 
Prepare the PCR Mix:
   1 tuben+1 tubesNEBNext‱ High-Fidelity 2X PCR Master Mix25 µl RP12 µl H2O (nuclease free)11 µl 
 
   

RP1 (Illumina RNA PCR Primer 1): 
5’ AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
53.1. Add 36 µL of the PCR Mix to each 10uL sample. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
53.2. To each sample, add 2 µL of one Illumina TruSeq Small RNA Index Adapter (RPI Series). When choosing indexes, keep in mind your sequencing arrangements (it's not advisable to have 2 samples with the same index, even if they come from different organisms).

 

Adaptor sequences (Illumina TruSeq Small RNA Index Adapter, RPI Series) are here :   
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
53.3. Incubate at 98 °C for 30 s, 
then run either 7 or 8 cycles of: 98 °C for 10 s, 60 °C for 30 s, 72 °C for 30 s; 
then 72 °C for 10 min, 
and finally hold at 4 °C
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
54. Bead Cleanup
Add 40 µL of undiluted, pre-warmed Ampure XP beads to each sample and incubate at Room temperature for 15 min.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
54.1. Move eppendorfs on a magnet rack and, once the beads have coalesced on the side of the tube and the supernatant is clear, remove the supernatant. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
54.2. Add 1 mL of 80% EtOH to each sample. Incubate for 30 s

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
54.3. Remove the supernatant. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
54.4. Repeat the 80% EtOH wash   
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
54.5. Dry up the beads (until they should lose their shimmer, up to  10 min. Drying time is influenced by the humidity. Over and under drying leads to material loss!!)
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
54.6. Resuspend in 25 µL of nuclease-free water (away from magnet). Let elute for ~ 2 min
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
54.7. Place sample in magnet rack, once the beads have coalesced on the side of the tube and the eluant is clear, transfer 25 µL of eluant to a new tube.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
55. Bead Cleanup
Add 20 µL of undiluted, pre-warmed Ampure XP beads to each sample and incubate at Room temperature for 15 min.
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
55.1. Move eppendorfs on a magnet rack and, once the beads have coalesced on the side of the tube and the supernatant is clear, remove the supernatant. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
55.2. Add 1 mL of 80% EtOH to each sample. Incubate for 30 s

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
55.3. Remove the supernatant. 
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
55.4. Repeat the 80% EtOH wash   
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
55.5. Dry up the beads (until they should lose their shimmer, up to  10 min. Drying time is influenced by the humidity. Over and under drying leads to material loss!!)
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
55.6. Resuspend in 10 µL of nuclease-free water (away from magnet). Let elute for ~ 2 min
SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
55.7. Place each sample in magnet rack. Once the beads have coalesced on the side of the tube and the eluant is clear, transfer 10 µL of eluant to new tubes. 
Label these tubes carefully for they contain the final library. Store at -20C if not planning on measuring concentration and size distribution soon, 

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
56. Measure the concentration with a Qubit High Sensitivity DNA assay. 
Measure the distribution of lengths of the library with a High Sensitivity DNA Bioanalyser. 
Store at -20C if not planning on sequencing soon.  

 

SECTION: DAY 5: rRNA depletion, RNA adapter ligation, RT, PCR and final library
57. Proceed to sequence each library at a depth of ~60M reads per plate. 
 

SECTION: Embryo Dissociation
2.1. ALTERNATIVE: Preparing Wash Buffers (not for CellTrace staining)
For those not planning to do any stainings, they only need to prepare the regular Wash Buffer following this recipe and the WB1 by adding EDTA to the regular WB.
           
  Wash
  Buffer 
  Concentration 
  Volume 50 mL
  Volume 10 mL
    
  H2O
  RNAse free water
   
  47.8 mL
  9.56 mL
  
  HEPES pH 7.5
  1M
  1 mL
  200 ml
  
  NaCl
  5M
  1.5 mL
  300 ml
  
  Spermidine solution
   
  3.6 µl
  0.72 ml
  
  Tween (10%)
  0.05%
  250 µl
  50 ml
  
  Protease inhibitor
   
  1 complete
  tablet
   
    
Store at 4 °C or in a cold room. Use for up to 2 days. 

WB1 
To an aliquot of WB, add 4 µL of 0.5 Molarity (M) EDTA for each 1 mL of WB. 
Store at 4 °C or in a cold room. Use for up to 2 days. 
We suggest not turning all of the regular WB in WB1 immediately, as the regular WB will still be used during day 2. 
SECTION: Embryo Dissociation
5.1. centrifuge at 600 rcf, 3 min with the swinging buckets centrifuge. 
SECTION: Embryo Dissociation
6.1. If using the Protease Solution, at the end of the dissociation, add 34uL of Protease Stop Solution for each 200uL of Protease Solution.