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   Mansour, Konstantinos Anestis, Fabrice Not, Uwe John

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">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.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">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.</div></div><div class = "text-block"><span>We report on transcriptomic methods that we have successfully performed on single cells of Acantharia, </span><span style = "font-style:italic;">Strombidium basimorphum, </span><span>and </span><span style = "font-style:italic;">Prymnesium parvum. </span></div><div class = "text-block"><span>This protocol follows up after total RNA extraction (from the protocol at </span><a href="https://dx.doi.org/10.17504/protocols.io.bp6xmrfn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">dx.doi.org/10.17504/protocols.io.bp6xmrfn</span></a><span>) 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).</span></div><div class = "text-block"><span>The subsequent cDNA library is prepared following the </span><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span>. </span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dwu536c6.jpg" /></div></div>

[STEPS]
?. [cDNA synthesis preparations]
Label for each sample a tube .
?. [cDNA synthesis preparations]
Prepare a 72°C incubator (e.g. a thermocycler)
?. [cDNA synthesis preparations]
Thaw other reagents  – except SmartScribe Reverse Transcriptase, take that from the freezer only once needed.
on ice
?. [cDNA synthesis preparations]
Thaw your RNA samples   (as prepared in dx.doi.org/10.17504/protocols.io.bp6xmrfn)
on ice
?. [cDNA synthesis preparations]
Prepare 10X Reaction Buffer (RB),  as follows  (1 µL is used per sample (adjust as needed, & write down exact volumes):
on ice
?. [cDNA synthesis preparations]
(from SMART-Seq4 kit)(white cap from SMART-Seq4 kit)
19 µl
1 µl
?. [cDNA synthesis]
Take into clean (labeled)  of RNA sample &  of RB(total 10.5 µL volume, adjust with depending on RNA sample)
[ to 9.5 ul]
1 µl
For single-cells we recommend  total RNA. In essence either all total RNA sample can be used, or it is safer to use
?. [cDNA synthesis]
Place samples  and add  of (blue cap) to the samples.
on ice
1 µl
We are performing 17+ PCR cycles. If fewer cycles are envisioned   of  should be used instead, though keeping the total volume the same by disregarding step 7.1).
?. [cDNA synthesis]
add  (total volume 12.5 µL)
1 µl
?. [cDNA synthesis]
Mix gently (vortex) & spin down
?. [cDNA synthesis]
Incubate samples at  for
72 °C
Immediately proceed to step 8 after incubation finishes
?. [cDNA synthesis]
While samples are incubating prepare Master Mix (MM) as below for each sample (+10%; write down exact volumes)
on ice
?. [cDNA synthesis]
(red cap)    (make sure precipitates are dissolved) (pink cap) (white cap)
4 µl
1 µl
5 µl
?. [cDNA synthesis]
Immediately after the 3 min 72°C incubation from step 8 put samples  for During this incubation time on ice perform steps 11 and 12.
on ice
?. [cDNA synthesis]
Preheat thermocycler to
42 °C
?. [cDNA synthesis]
Take the  (purple cap), gently mix it without vortexing and add to the prepared Master Mix (from step 9):
?. [cDNA synthesis]
for each sample (x #samples +10%)
2 µl
?. [cDNA synthesis]
Mix MM by gentle vortex and spin down
?. [cDNA synthesis]
Add  of the MM to the samples (total volume now 20 µL)
7.5 µl
?. [cDNA synthesis]
Mix by pipetting and follow with short spindown
?. [cDNA synthesis]
Incubate samples in pre-heated Thermocyler with heated lid and the following program:,;
42 °C
70 °C
[forever]
?. [cDNA synthesis]
STOPPING POINT - 4°C overnight
?. [cDNA Amplification]
Thaw all the reagents (see step 18)  except the enzyme(Vortex and spin down reagents except for enzyme)
on ice
?. [cDNA Amplification]
Preheat thermocycler to
95 °C
?. [cDNA Amplification]
Prepare Mastermix (+10%), one sample is as below:
?. [cDNA Amplification]
(green cap) (take out last minute and mix without vortexing, spin down)
25 µl
1 µl
3 µl
1 µl
?. [cDNA Amplification]
Add  of Mastermix to each sample from cDNA synthesis.Mix well (pipetting) and spin down gently.
30 µl
?. [cDNA Amplification]
Run samples on pre-heated thermocycler with the program:  ABC1  95°C    1 min       2  98°C    10 sec  repeat step 2, 18 times3  65°C    30 sec  4  68°C    3 min  5  72°C    10 min       6  4°C    forever
ABC1  95°C    1 min       2  98°C    10 sec  repeat step 2, 18 times3  65°C    30 sec  4  68°C    3 min  5  72°C    10 min       6  4°C    forever
This thermocycler program is run with 18 cycles and works for us. Nonetheless, it is recommended to test this beforehand. Over-amplification can result in ahigher yield of cDNA, however, it introduces a bias towards more abundant transcripts. We settled on the following number of amplification cycles.  ABC1  Species    cDNA kit    Number of cycles  2  Strombidium basimorphum    SMARTseq-v4    18  3  Prymnesium parvum    NEBNext    25  4  Acantharia    SMARTseq-v4    18  5  Acantharia    NEBNext    16
?. [cDNA Amplification]
STOPPING POINT 4°C overnight
?. [cDNA cleanup/bead purification]
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,  x samples (plus extra)Bring the bead aliquot to for at least Vortex the bead aliquot until evenly mixedPrepare fresh 80% EtOH, 400 µL x samples
22.5 µl
4 Room temperature
?. [cDNA cleanup/bead purification]
Add  of beads to each sample (amplified cDNA from the previous section)
22.5 µl
?. [cDNA cleanup/bead purification]
Mix by pipetting up and down at least 10 times, and vortex
?. [cDNA cleanup/bead purification]
Incubate at  to let cDNA bind to the beads
4 Room temperature
?. [cDNA cleanup/bead purification]
Briefly spin down and place the samples on a  for  or longer. Until the liquid appears completely clear and there are no beads in the supernatant.
?. [cDNA cleanup/bead purification]
Pipet and discard the supernatant (72.5 µL), keeping the samples in the magnetic device
?. [cDNA cleanup/bead purification]
Keeping the samples in the magnetic device, add fresh  to each sample.
200 µl
Do not disturb the beads
?. [cDNA cleanup/bead purification]
Wait
?. [cDNA cleanup/bead purification]
Pipet and discard supernatant containing contaminants (use 100 µL)
?. [cDNA cleanup/bead purification]
Repeat the EtOH washing step for a total of 2 washing steps
?. [cDNA cleanup/bead purification]
Briefly spin the samples to collect liquid off the sides
?. [cDNA cleanup/bead purification]
Place samples back in the magnetic device for, beads will again be collected on the side
?. [cDNA cleanup/bead purification]
Remove all remaining ethanol/supernatant with a pipet (use 10 µL pipet)
?. [cDNA cleanup/bead purification]
Place samples at for minutes. (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.
4 Room temperature
?. [cDNA cleanup/bead purification]
Once the beads are dry add  of Elution buffer to all samples to cover the bead pellet
15 µl
?. [cDNA cleanup/bead purification]
Remove samples from the magnetic device
?. [cDNA cleanup/bead purification]
Mix to re-suspend the beads by (multi)pipetting (can scrap of beads from the side)
?. [cDNA cleanup/bead purification]
Incubate at for (longer) to rehydrate
4 Room temperature
?. [cDNA cleanup/bead purification]
Briefly spin the samples to collect liquid off the sides
?. [cDNA cleanup/bead purification]
Place the samples back in the magnetic device for , until the solution is completely clear
?. [cDNA cleanup/bead purification]
Transfer the clear supernatant containing purified cDNA to   tube (use 10 µL pipet).
Beads that do not pellet can be pipetted for resuspension and then towards the magnet, and incubation continued until there are no more beads in the supernatant
?. [cDNA cleanup/bead purification]
Make immediately an aliquot for Bioanalyzer analysis to prevent unnecessary freeze-thawing cycles.
?. [cDNA cleanup/bead purification]
STOPPING POINT - Label and store at
-20 °C
?. [cDNA Sample verification]
Check the quality of cDNA by  following the manufacture's protocol.
?. [cDNA Sample verification]
Quantify and calculate the concentration of cDNA. This is needed for the next cDNA library procedure.
?. [cDNA library preparation and indexing – Nextera XT]
Proceed with cDNA library preparation only for good quality samples from the previous step.
?. [cDNA library preparation and indexing – Nextera XT]
Normalize cDNA samples to 30pg/ulDilute 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.
?. [cDNA library preparation and indexing – Nextera XT]
Prepare to work very timely for this protocolPreheat a PCR thermocycler to , with preheat lid at 100 °CPrepare 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
55 °C
?. [cDNA library preparation and indexing – Nextera XT]
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).
?. [cDNA library preparation and indexing – Nextera XT]
Changes to manufacturer' s protocol:Start the tagmentation with   of 30 pg/µl amplified cDNA sample (from step 37)
5 µl
?. [cDNA library preparation and indexing – Nextera XT]
Store samples at   for up to 2 days or proceed immediately with purification
4 °C
?. [cDNA library purification]
Preparations:Vortex the bead stock well ( ), this needs to be very well and evenly mixed Aliquot beads,  x samples (plus extra)Bring the bead aliquot to for at least Vortex the bead aliquot until evenly mixedPrepare fresh 80% EtOH, 400 µL x #samples
30 µl
4 Room temperature
?. [cDNA library purification]
Spin down your indexed cDNA samples (total 50 µL)
?. [cDNA library purification]
Add 30 µL of to each sampleShake/vortex for
?. [cDNA library purification]
Incubate at  to let cDNA bind to the beads
4 Room temperature
?. [cDNA library purification]
Briefly spin down and place the samples on a  for  or longer. Until the liquid appears completely clear and there are no beads in the supernatant.
?. [cDNA library purification]
Pipet and discard the supernatant (80 µL), keeping the samples in the magnetic device
?. [cDNA library purification]
Keeping the samples in the magnetic device, add fresh  to each sample.
200 µl
Do not disturb the beads
?. [cDNA library purification]
Wait
?. [cDNA library purification]
Pipet and discard supernatant containing contaminants (use 100 µL pipet)
?. [cDNA library purification]
Repeat the EtOH washing step for a total of 2 washing steps
?. [cDNA library purification]
Briefly spin the samples to collect liquid off the sides
?. [cDNA library purification]
Place samples back in the magnetic device for, beads will again be collected on the side
?. [cDNA library purification]
Remove all remaining ethanol/supernatant with a pipet (use 10 µL pipet)
?. [cDNA library purification]
Place samples at for minutes. Until the pellet is no longer shiny, but before a crack appears. It needs to be ‘just’ dry, matte with no shine.
4 Room temperature
?. [cDNA library purification]
Once the beads are dry add  of (NexteraXT kit) to all samples to cover the bead pellet
52.5 µl
?. [cDNA library purification]
Remove samples from the magnetic device
?. [cDNA library purification]
Mix to re-suspend the beads by (multi)pipetting (can scrap of beads from the side)
?. [cDNA library purification]
Vortex for   followed by a very short spindown
?. [cDNA library purification]
Incubate at for  to rehydrate
4 Room temperature
?. [cDNA library purification]
Briefly spin the samples to collect liquid off the sides
?. [cDNA library purification]
Place the samples back in the magnetic device for , until the solution is completely clear
?. [cDNA library purification]
Transfer the clear supernatant (50 uL) containing your purified cDNA library to   tube (use 10 µL pipet).
Beads that do not pellet can be pipetted for resuspension and then towards the magnet, and incubation continued until there are no more beads in the supernatant
?. [cDNA library purification]
Make immediately an aliquot for Bioanalyser analysis to prevent unnecessary freeze-thawing cycles.
?. [cDNA library purification]
STOPPING POINT - Label and store at  for sequencing
-20 °C
?. [cDNA library verification]
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.
?. [cDNA library verification]
Quantify and calculate the concentration of cDNA by smear analysis. This is needed for the normalization of samples for sequencing.
?. [4.4.3	Follow up steps: library quality control; sample normalization/dilution and pooling for sequencing]
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.