TITLE: cDNA library preparation from total RNA extracts of Single-cell marine protists (e.g. Acantharia, Strombidium basimorphum, and Prymnesium parvum) for transcriptome sequencing
AUTHORS: Joost S Mansour, Konstantinos  Anestis, Fabrice  Not, Uwe John

[DESCRIPTION]
Many marine protists are not culturable and therefore challenging to study, nonetheless, they are essential in all marine ecosystems. The development of single-cell techniques is allowing for more marine protists to be studied. Such genomic approaches aim to help to disentangle heterotrophic processes such as phagotrophy from osmotrophy and phototrophic-induced anabolic activities. This information will then support cellular and metabolic modeling by better elucidating the physiological mechanisms and quantifying their importance in different scenarios.
However, single-cell protocols and low input RNA kits for transcriptomics are usually made for and tested with mammalian cells, as such the feasibility and efficiency of single-cell transcriptomics on highly diverse mixotrophic protists is not always known. Often single-cell transcriptomics of microbial eukaryotes shows low transcript recovery rates and large variability.

We report on transcriptomic methods that we have successfully performed on single cells of Acantharia, Strombidium basimorphum, and Prymnesium parvum. 
This protocol follows up after total RNA extraction (from the protocol at dx.doi.org/10.17504/protocols.io.bp6xmrfn) to prepare cDNA libraries for Illumina sequencing. The described protocol uses the SMART-Seq4 kit (Takara #634891) for cDNA synthesis and amplification, but this can also be successfully performed with the NEBNext kit (NEB #E6421). The NEBNext kit protocol is very similar to the protocol described here and generally the manufacture's protocol can be followed but see the notes at step 4 and step 18 of this protocol, and do the final elution after cDNA purification in 10 mM Tris (pH 8.0).
The subsequent cDNA library is prepared following the  . 

  


[BEFORE_START]
Total RNA needs to have been extracted (Protocol: dx.doi.org/10.17504/protocols.io.bp6xmrfn) and when possible quantified and quality checked by Bioanalyzer. If Bioanalyzer analysis was possible, only continue with good quality RNA extracts.


Thaw reagents (except enzymes).
Allow reagents that need to be at room temperature to incubate at Room temperature (i.e.   and GC nucleic acids purification beads.
Set thermocycler programs and pre-heat thermocyclers.
For the cDNA purification step Prepare fresh 80% ethanol from   with   


[GUIDELINES]
Always wear clean RNase-free gloves. 
Clean workspace (and thermocyclers) with ethanol and an RNase Decontamination Solution.
Work on ice 
If possible use a dedicated set of pipettes for RNA and use filter tips.

[STEPS]
SECTION: cDNA synthesis preparations
1. Label for each sample a tube  .

SECTION: cDNA synthesis preparations
2. Prepare a 72°C incubator (e.g. a thermocycler)
SECTION: cDNA synthesis preparations
3. Thaw other reagents on ice – except SmartScribe Reverse Transcriptase, take that from the freezer only once needed.

SECTION: cDNA synthesis preparations
4. Thaw your RNA samples on ice  (as prepared in dx.doi.org/10.17504/protocols.io.bp6xmrfn)

SECTION: cDNA synthesis preparations
5. Prepare 10X Reaction Buffer (RB), on ice as follows  (1 µL is used per sample (adjust as needed, & write down exact volumes):
SECTION: cDNA synthesis preparations
5.1. 19 µL   (from SMART-Seq4 kit)
1 µL (white cap from SMART-Seq4 kit)
Mix/vortex and spin down (avoid bubbles)
SECTION: cDNA synthesis
6. Take into clean (labeled)   1 µL of RNA sample & 1 µL of RB
(total 10.5 µL volume, adjust with  depending on RNA sample)
 

SECTION: cDNA synthesis
7. Place samples on ice and add 1 µL of  (blue cap) to the samples.
 

SECTION: cDNA synthesis
7.1. add 1 µL   (total volume 12.5 µL)

SECTION: cDNA synthesis
7.2. Mix gently (vortex) & spin down
SECTION: cDNA synthesis
8. Incubate samples at 72 °C for 3 min

 

SECTION: cDNA synthesis
9. While samples are incubating prepare Master Mix (MM) as below for each sample (+10%; write down exact volumes) on ice

SECTION: cDNA synthesis
9.1. 4 µL   (red cap)
    (make sure precipitates are dissolved)
1 µL   (pink cap)
0.5 µL   (white cap)
SECTION: cDNA synthesis
10. Immediately after the 3 min 72°C incubation from step 8 put samples on ice for 2 min
During this incubation time on ice perform steps 11 and 12.
SECTION: cDNA synthesis
11. Preheat thermocycler to 42 °C

SECTION: cDNA synthesis
12. Take the   (purple cap), gently mix it without vortexing and add to the prepared Master Mix (from step 9):

SECTION: cDNA synthesis
12.1.  2 µL   for each sample (x #samples +10%)

SECTION: cDNA synthesis
12.2.  Mix MM by gentle vortex and spin down
SECTION: cDNA synthesis
13. Add 7.5 µL of the MM to the samples (total volume now 20 µL)

SECTION: cDNA synthesis
13.1. Mix by pipetting and follow with short spindown
SECTION: cDNA synthesis
14. Incubate samples in pre-heated Thermocyler with heated lid and the following program:
 42 °C 90 min,
70 °C 10 min; 
4 °C 


SECTION: cDNA synthesis
15. STOPPING POINT - 4°C overnight
SECTION: cDNA Amplification
16. Thaw all the reagents (see step 18) on ice except the enzyme
(Vortex and spin down reagents except for enzyme)
SECTION: cDNA Amplification
17. Preheat thermocycler to 95 °C
SECTION: cDNA Amplification
18. Prepare Mastermix (+10%), one sample is as below:
SECTION: cDNA Amplification
18.1. 

25 µL  
1 µL  (green cap)
3 µL  
1 µL   (take out last minute and mix without vortexing, spin down)

Mix Master Mix well and gently (finger flick) and spin down

SECTION: cDNA Amplification
19. Add 30 µL of Mastermix to each sample from cDNA synthesis.
Mix well (pipetting) and spin down gently.

SECTION: cDNA Amplification
20. Run samples on pre-heated thermocycler with the program:
 
 
     95°C      1 min               98°C      10 sec      18 cycles        65°C      30 sec        68°C      3 min        72°C      10 min               4°C      forever          

 




SECTION: cDNA Amplification
21. STOPPING POINT 4°C overnight
SECTION: cDNA cleanup/bead purification
22. Preparations:
Label for each sample two tubes  . One tube is used for the cDNA after purification, and one is for an aliquot of the purified cDNA for Bioanalyzer.
Vortex the bead stock well (  ), this needs to be very well and evenly mixed 
Aliquot beads, 22.5 µL x samples (plus extra)
Bring the bead aliquot to 4 Room temperaturefor at least 30 min
Vortex the bead aliquot until evenly mixed
Prepare fresh 80% EtOH, 400 µL x samples


SECTION: cDNA cleanup/bead purification
23. Add 22.5 µL of beads to each sample (amplified cDNA from the previous section)


SECTION: cDNA cleanup/bead purification
23.1. Mix by pipetting up and down at least 10 times, and vortex
SECTION: cDNA cleanup/bead purification
24. Incubate at 4 Room temperature8 min to let cDNA bind to the beads

SECTION: cDNA cleanup/bead purification
25. Briefly spin down and place the samples on a   for 5 min or longer. Until the liquid appears completely clear and there are no beads in the supernatant.
 


SECTION: cDNA cleanup/bead purification
26. Pipet and discard the supernatant (72.5 µL), keeping the samples in the magnetic device
SECTION: cDNA cleanup/bead purification
27. Keeping the samples in the magnetic device, add 200 µLfresh   to each sample.
 

SECTION: cDNA cleanup/bead purification
27.1. Wait 30 s

SECTION: cDNA cleanup/bead purification
27.2. Pipet and discard supernatant containing contaminants (use 100 µL)
SECTION: cDNA cleanup/bead purification
28. Repeat the EtOH washing step for a total of 2 washing steps   
SECTION: cDNA cleanup/bead purification
29. Briefly spin the samples to collect liquid off the sides
SECTION: cDNA cleanup/bead purification
30. Place samples back in the magnetic device for30 s, beads will again be collected on the side

SECTION: cDNA cleanup/bead purification
31. Remove all remaining ethanol/supernatant with a pipet (use 10 µL pipet)
SECTION: cDNA cleanup/bead purification
32. Place samples at 4 Room temperaturefor 2 minminutes. (it might take a bit longer)
Until the pellet is no longer shiny, but before a crack appears. It needs to be ‘just’ dry, matte with no shine. 

SECTION: cDNA cleanup/bead purification
33. Once the beads are dry add 15 µL of Elution buffer to all samples to cover the bead pellet

SECTION: cDNA cleanup/bead purification
33.1. Remove samples from the magnetic device
SECTION: cDNA cleanup/bead purification
33.2. Mix to re-suspend the beads by (multi)pipetting (can scrap of beads from the side)
SECTION: cDNA cleanup/bead purification
34. Incubate at 4 Room temperaturefor2 min (longer) to rehydrate 

SECTION: cDNA cleanup/bead purification
35. Briefly spin the samples to collect liquid off the sides
SECTION: cDNA cleanup/bead purification
36. Place the samples back in the magnetic device for 1 min, until the solution is completely clear

SECTION: cDNA cleanup/bead purification
37. 
Transfer the clear supernatant containing purified cDNA to    tube (use 10 µL pipet).
 


SECTION: cDNA cleanup/bead purification
37.1. Make immediately an aliquot for Bioanalyzer analysis to prevent unnecessary freeze-thawing cycles.
SECTION: cDNA cleanup/bead purification
38. STOPPING POINT - Label and store at -20 °C
SECTION: cDNA Sample verification
39. Check the quality of cDNA by   following the manufacture's protocol. 

 

SECTION: cDNA Sample verification
39.1. Quantify and calculate the concentration of cDNA. This is needed for the next cDNA library procedure.
SECTION: cDNA library preparation and indexing – Nextera XT
40. Proceed with cDNA library preparation only for good quality samples from the previous step.
SECTION: cDNA library preparation and indexing – Nextera XT
41. Normalize cDNA samples to 30pg/ul

Dilute each sample of amplified and purified cDNA to 30 pg/µL in either Elution buffer or as per the final step of the used protocol for cDNA purification. Work with a minimum of 1 µL amplified cDNA and a total volume of 5 µL.

SECTION: cDNA library preparation and indexing – Nextera XT
42. Prepare to work very timely for this protocol
Preheat a PCR thermocycler to 55 °C, with preheat lid at 100 °C
Prepare from the   the ATM and NT reagents in sufficient quantity (i.e. 5 ul per sample for each) separated over multiple tubes to facilitate multi-pipetting


SECTION: cDNA library preparation and indexing – Nextera XT
43. Follow the    manufacturer's protocol for  " Tagment genomic DNA", and "Amplify Libraries", with the changes listed below.

Refer to pages 7-9 of the Nextera XT manual (https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera-xt/nextera-xt-library-prep-reference-guide-15031942-05.pdf).
SECTION: cDNA library preparation and indexing – Nextera XT
44. Changes to manufacturer' s protocol:
Start the tagmentation with 5 µL  of 30 pg/µl amplified cDNA sample (from step 37)
all steps indicated as "centrifuge at 280 x g at 20 °C for 1 minute" can be substituted short spindown in a tabletop mini-centrifuge.
SECTION: cDNA library preparation and indexing – Nextera XT
45. Store samples at 4 °C  for up to 2 days or proceed immediately with purification

SECTION: cDNA library purification
46. Preparations:
Vortex the bead stock well (  ), this needs to be very well and evenly mixed 
Aliquot beads, 30 µL x samples (plus extra)
Bring the bead aliquot to 4 Room temperaturefor at least 30 min
Vortex the bead aliquot until evenly mixed
Prepare fresh 80% EtOH, 400 µL x #samples
SECTION: cDNA library purification
47. Spin down your indexed cDNA samples (total 50 µL)
SECTION: cDNA library purification
48. Add 30 µL of  to each sample
Mix by pipetting up and down
Shake/vortex for2 min

SECTION: cDNA library purification
49. Incubate at 4 Room temperature5 min to let cDNA bind to the beads

SECTION: cDNA library purification
50. Briefly spin down and place the samples on a   for 5 min or longer. Until the liquid appears completely clear and there are no beads in the supernatant.

SECTION: cDNA library purification
51. Pipet and discard the supernatant (80 µL), keeping the samples in the magnetic device
SECTION: cDNA library purification
52. Keeping the samples in the magnetic device, add 200 µLfresh   to each sample.
 

SECTION: cDNA library purification
52.1. Wait 30 s

SECTION: cDNA library purification
52.2. Pipet and discard supernatant containing contaminants (use 100 µL pipet)
SECTION: cDNA library purification
53. Repeat the EtOH washing step for a total of 2 washing steps   
SECTION: cDNA library purification
54. Briefly spin the samples to collect liquid off the sides
SECTION: cDNA library purification
55. Place samples back in the magnetic device for30 s, beads will again be collected on the side

SECTION: cDNA library purification
56. Remove all remaining ethanol/supernatant with a pipet (use 10 µL pipet)
SECTION: cDNA library purification
57. Place samples at 4 Room temperaturefor 5 minminutes. 
Until the pellet is no longer shiny, but before a crack appears. It needs to be ‘just’ dry, matte with no shine. 

SECTION: cDNA library purification
58. Once the beads are dry add 52.5 µL of  (NexteraXT kit) to all samples to cover the bead pellet

SECTION: cDNA library purification
58.1. Remove samples from the magnetic device
SECTION: cDNA library purification
58.2. Mix to re-suspend the beads by (multi)pipetting (can scrap of beads from the side)
SECTION: cDNA library purification
58.3. Vortex for 2 min  followed by a very short spindown

SECTION: cDNA library purification
59. Incubate at 4 Room temperaturefor2 min  to rehydrate 

SECTION: cDNA library purification
60. Briefly spin the samples to collect liquid off the sides
SECTION: cDNA library purification
61. Place the samples back in the magnetic device for 2 min, until the solution is completely clear

SECTION: cDNA library purification
62. 
Transfer the clear supernatant (50 uL) containing your purified cDNA library to    tube (use 10 µL pipet).
 

SECTION: cDNA library purification
62.1. Make immediately an aliquot for Bioanalyser analysis to prevent unnecessary freeze-thawing cycles.
SECTION: cDNA library purification
63. STOPPING POINT - Label and store at -20 °C for sequencing
SECTION: cDNA library verification
64. Check the quality of the cDNA libraries by   following the manufacture's protocol. Alternatively, a  Bioanalyser DNA 7500 Kit (Agilent #5067-1506) could be used as a more cost-efficient alternative and if sample concentration permitting. See for example the third graph.

 
  
  

SECTION: cDNA library verification
64.1. Quantify and calculate the concentration of cDNA by smear analysis. This is needed for the normalization of samples for sequencing.
SECTION: 4.4.3	Follow up steps: library quality control; sample normalization/dilution and pooling for sequencing
65. The quality and quantity control of the generated cDNA libraries is performed using the Agilent High Sensitivity DNA kit (Agilent #5067-4626). In case primer-dimers or adapters are still present, an additional step of cleaning with magnetic beads is to be performed. A bead to sample ratio of 0.7:1 was found to be efficient in eliminating both primer dimers and remaining adapters. 

The cDNA libraries are normalized to equal molarity, as well as fragment size before the final pooling and subsequent sequencing. Calculate nM cDNA of each sample as: nM DNA = [ng/µL] x 106 / (660 x fragment length bp). Where the concentration in ng/µL and the average fragment length in base pairs are obtained from Bioanalyzer smear analysis.

The molarity upon which the cDNA libraries are normalized is determined based on the yield of cDNA, as well as the requirements for the subsequent sequencing (e.g. >0.5 nM). The final pool of all the samples should again be checked using the Bioanalyzer in order to verify that the normalization process was successful.

The pools are ready for Illumina sequencing.