Jan 05, 2022
Version 3
cDNA library preparation from total RNA extracts of Single-cell marine protists (e.g. Acantharia, Strombidium basimorphum, and Prymnesium parvum) for transcriptome sequencing V.3
- Joost S Mansour1,
- Konstantinos Anestis2,
- Fabrice Not3,
- Uwe John2
- 1University of Arizona;
- 2Alfred-Wegener-Institute Helmholtz Centre for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany;
- 3CNRS & Sorbonne University - Station Biologique de Roscoff
- Ecology of Marine Plankton (ECOMAP) team - RoscoffTech. support email: phytopk@sb-roscoff.fr
Protocol Citation: Joost S Mansour, Konstantinos Anestis, Fabrice Not, Uwe John 2022. cDNA library preparation from total RNA extracts of Single-cell marine protists (e.g. Acantharia, Strombidium basimorphum, and Prymnesium parvum) for transcriptome sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.b3ifqkbnVersion created by Joost S Mansour
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 05, 2022
Last Modified: January 05, 2022
Protocol Integer ID: 56615
Keywords: Single-cell, Acantharia, RNA, transcriptomics, cDNA library, Strombidium, Radiolaria, ciliates, RNA-seq,
Funders Acknowledgements:
MixITiN - European Union's Horizon 2020, Marie Skłodowska-Curie
Grant ID: 766327
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Abstract
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 Nextera XT DNA Library Preparation KitilluminaCatalog #FC-131-1096 .
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.
Materials
cDNA synthesis
- PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230 (x #samples)
- 1.5 ml reaction tubeEppendorf (x2 for mastermix and reaction buffer)
- Ice
- nuclease free water
- 10X Lysis Buffer TakaraCatalog #634888
- RNase InhibitorTakaraCatalog #2313A
- SMART-seq v4 Oligonucleotide (46 μM)TakarabioCatalog #634888 (48 µM, 1 µL per sample)
- 5X Ultra Low First-Strand Buffer TakarabioCatalog #634888 (4 µL per sample)
- SMART-seq CDS Primer II A (12 μM)TakarabioCatalog #634888 (1 µL per sample)
- SMARTScribe Reverse TranscriptaseTakarabioCatalog #634888 (2 µL per sample)
cDNA amplification
- 1.5 ml reaction tubeEppendorf (for mastermix)
- nuclease free water (3 µL per sample)
- 2X SeqAmp PCR BufferTakaraCatalog #638526 (25 µL per sample)
- SeqAmp DNA PolymeraseTakaraCatalog #638504 (1 µL per sample)
- PCR Primer II A (12 μM)TakaraCatalog #634888 (1 µL per sample)
cDNA purification
- 80% Ethanol (made from 100% Molecular grade ethanol and nuclease free water ) (400 µL per sample)
- 50 mL Falcon Tubes (for 80% ethanol)
- AMPure XP Beads (this can be substituted for a similar product, we use CleanNGS (GC Biotech, CNGS-0050)
- Magnetic Stand-96ThermofisherCatalog #AM10027
- PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230
- PCR Tubes, 0.2mL, flat cap, natural, PCR Tube; 0.2mL; Natural; w/flat cap; 1000/Pk.Thermo FisherCatalog #3412 (x #samples x2)
- 1.5 ml reaction tubeEppendorf (for bead aliquot, a 5 mL tube might be preferred)
cDNA library preparation, indexing, and purification
- PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230 (2x for reagent aliquots)
- PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230 (2x #samples)
- Nextera XT DNA Library Preparation KitilluminaCatalog #FC-131-1096
- Nextera XT Index Kit v2 Set A (96 indexes 384 samples)illuminaCatalog #FC-131-2001
- AMPure XP Beads (This can be substituted for a similar product, we use CleanNGS (GC Biotech, CNGS-0050).
- 80% Ethanol (made from 100% Molecular grade ethanol and nuclease free water ) (400 µL per sample)
- 1.5 ml reaction tubeEppendorf (for bead aliquot, a 5 mL tube might be preferred)
- Magnetic Stand-96ThermofisherCatalog #AM10027
General lab equipment
- Micropipettes and filter tips
- Vortex
- PCR Thermocycler
- Ice
Equipment
Mini-centrifuge
NAME
Centrifuge
TYPE
Fisher
BRAND
S67601B
SKU
LINK
Any standard mini centrifuge with adapters for different tube sizes will suffice
SPECIFICATIONS
Equipment
Bioanalyzer
NAME
Bioanalyzer
TYPE
Agilent
BRAND
G2991AA
SKU
LINK
Any bioanalyzer will suffice.
SPECIFICATIONS
Agilent High Sensitivity DNA Kit Agilent TechnologiesCatalog #5067-4626
Protocol materials
5X Ultra Low First-Strand Buffer Takara Bio Inc.Catalog #634888
100% Molecular grade ethanol
nuclease free water
Nextera XT DNA Library Preparation KitIllumina, Inc.Catalog #FC-131-1096
nuclease free water
2X SeqAmp PCR BufferTakara Bio Inc.Catalog #638526
80% Ethanol
nuclease free water
100% Molecular grade ethanol
Ice
PCR Primer II A (12 μM)Takara Bio Inc.Catalog #634888
nuclease free water
PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230
AMPure XP Beads
Magnetic Stand-96ThermofisherCatalog #AM10027
Vortex
RNase InhibitorTakara Bio Inc.Catalog #2313A
SMARTScribe Reverse TranscriptaseTakara Bio Inc.Catalog #634888
AMPure XP Beads
Magnetic Stand-96ThermofisherCatalog #AM10027
1.5 ml reaction tubeEppendorf
PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230
SeqAmp DNA PolymeraseTakara Bio Inc.Catalog #638504
100% Molecular grade ethanol
50 mL Falcon Tubes
Nextera XT DNA Library Preparation KitIllumina, Inc.Catalog #FC-131-1096
SMART-seq v4 Oligonucleotide (46 μM)Takara Bio Inc.Catalog #634888
SMART-seq CDS Primer II A (12 μM)Takara Bio Inc.Catalog #634888
PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230
PCR Thermocycler
Agilent High Sensitivity DNA Kit Agilent TechnologiesCatalog #5067-4626
Ice
PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230
PCR Tubes, 0.2mL, flat cap, natural, PCR Tube; 0.2mL; Natural; w/flat cap; 1000/Pk.Thermo FisherCatalog #3412
10X Lysis Buffer Takara Bio Inc.Catalog #634888
1.5 ml reaction tubeEppendorf
Nextera XT Index Kit v2 Set A (96 indexes 384 samples)Illumina, Inc.Catalog #FC-131-2001
nuclease free water
1.5 ml reaction tubeEppendorf
80% Ethanol
1.5 ml reaction tubeEppendorf
5X Ultra Low First-Strand Buffer Takara Bio Inc.Catalog #634888
PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230
10X Lysis Buffer Takara Bio Inc.Catalog #634888
RNase InhibitorTakara Bio Inc.Catalog #2313A
PCR Tubes, 0.2mL, flat cap, natural, PCR Tube; 0.2mL; Natural; w/flat cap; 1000/Pk.Thermo FisherCatalog #3412
nuclease free water
SMART-seq CDS Primer II A (12 μM)Takara Bio Inc.Catalog #634888
nuclease free water
SMART-seq v4 Oligonucleotide (46 μM)Takara Bio Inc.Catalog #634888
RNase InhibitorTakara Bio Inc.Catalog #2313A
5X Ultra Low First-Strand Buffer Takara Bio Inc.Catalog #634888
SMARTScribe Reverse TranscriptaseTakara Bio Inc.Catalog #634888
SMARTScribe Reverse TranscriptaseTakara Bio Inc.Catalog #634888
PCR Primer II A (12 μM)Takara Bio Inc.Catalog #634888
nuclease free water
SeqAmp DNA PolymeraseTakara Bio Inc.Catalog #638504
2X SeqAmp PCR BufferTakara Bio Inc.Catalog #638526
PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230
AMPure XP Beads
Magnetic Stand-96ThermofisherCatalog #AM10027
80% Ethanol
PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230
Agilent High Sensitivity DNA Kit Agilent TechnologiesCatalog #5067-4626
Nextera XT DNA Library Preparation KitIllumina, Inc.Catalog #FC-131-1096
Nextera XT DNA Library Preparation KitIllumina, Inc.Catalog #FC-131-1096
AMPure XP Beads
AMPure XP Beads
Magnetic Stand-96ThermofisherCatalog #AM10027
80% Ethanol
Resuspension Buffer
PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230
Agilent High Sensitivity DNA Kit Agilent TechnologiesCatalog #5067-4626
Safety warnings
We have tested this for work to acquire transcriptomes from Acantharia, Strombidinium basimorphum, and Prymnesium parvum.
Adhere to PPE, as dictated under local Health & Safety regulations.
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. 5X Ultra Low First-Strand Buffer TakarabioCatalog #634888 and GC nucleic acids purification beads.
- Set thermocycler programs and pre-heat thermocyclers.
- For the cDNA purification step Prepare fresh 80% ethanol from 100% Molecular grade ethanol with nuclease free water
cDNA synthesis preparations
cDNA synthesis preparations
Label for each sample a tube PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Takara Bio Inc.Catalog #AM12230 .
Prepare a 72°C incubator (e.g. a thermocycler)
Thaw other reagents On ice – except SmartScribe Reverse Transcriptase, take that from the freezer only once needed.
Prepare 10X Reaction Buffer (RB), On ice as follows (1 µL is used per sample (adjust as needed, & write down exact volumes):
- 19 µL 10X Lysis Buffer Takara Bio Inc.Catalog #634888 (from SMART-Seq4 kit)
- 1 µL RNase InhibitorTakara Bio Inc.Catalog #2313A (white cap from SMART-Seq4 kit)
- Mix/vortex and spin down (avoid bubbles)
cDNA synthesis
cDNA synthesis
1h 45m
1h 45m
Take into clean (labeled) PCR Tubes, 0.2mL, flat cap, natural, PCR Tube; 0.2mL; Natural; w/flat cap; 1000/Pk.Takara Bio Inc.Catalog #3412 1 µL to 9.5 ul of RNA sample & 1 µL of RB
(total 10.5 µL volume, adjust withnuclease free waterTakara Bio Inc. depending on RNA sample)
Note
For single-cells we recommend 5 µL total RNA. In essence either all total RNA sample can be used, or it is safer to use <50% to allow redo when needed and [RNA] permitting. The total amplification cycles would also be affected by the volume used here.
Place samples On ice and add 1 µL of SMART-seq CDS Primer II A (12 μM)Takara Bio Inc.Catalog #634888 (blue cap) to the samples.
Note
We are performing 17+ PCR cycles. If fewer cycles are envisioned 2 µL of SMART-seq CDS Primer II A (12 μM)TakarabioCatalog #634888 should be used instead, though keeping the total volume the same by disregarding step 7.1).
add 1 µL nuclease free waterTakara Bio Inc. (total volume 12.5 µL)
Mix gently (vortex) & spin down
Incubate samples at 72 °C for 00:03:00
Note
Immediately proceed to step 8 after incubation finishes
3m
While samples are incubating prepare Master Mix (MM) as below for each sample (+10%; write down exact volumes) On ice
- 4 µL 5X Ultra Low First-Strand Buffer Takara Bio Inc.Catalog #634888 (red cap)
(make sure precipitates are dissolved)
- 1 µL SMART-seq v4 Oligonucleotide (46 μM)Takara Bio Inc.Catalog #634888 (pink cap)
- 0.5 µL RNase InhibitorTakara Bio Inc.Catalog #2313A (white cap)
Immediately after the 3 min 72°C incubation from step 8 put samples On ice for 00:02:00
During this incubation time on ice perform steps 11 and 12.
2m
Preheat thermocycler to 42 °C
Take the SMARTScribe Reverse TranscriptaseTakara Bio Inc.Catalog #634888 (purple cap), gently mix it without vortexing and add to the prepared Master Mix (from step 9):
2 µL SMARTScribe Reverse TranscriptaseTakara Bio Inc.Catalog #634888 for each sample (x #samples +10%)
Mix MM by gentle vortex and spin down
Add 7.5 µL of the MM to the samples (total volume now 20 µL)
Mix by pipetting and follow with short spindown
Incubate samples in pre-heated Thermocyler with heated lid and the following program:
42 °C 01:30:00 ,
70 °C 00:10:00 ;
4 °C forever
1h 40m
STOPPING POINT - 4°C overnight
cDNA Amplification
cDNA Amplification
Thaw all the reagents (see step 18) On ice except the enzyme
(Vortex and spin down reagents except for enzyme)
Preheat thermocycler to 95 °C
Prepare Mastermix (+10%), one sample is as below:
- 25 µL 2X SeqAmp PCR BufferTakara Bio Inc.Catalog #638526
- 1 µL PCR Primer II A (12 μM)Takara Bio Inc.Catalog #634888 (green cap)
- 3 µL nuclease free waterTakara Bio Inc.
- 1 µL SeqAmp DNA PolymeraseTakara Bio Inc.Catalog #638504 (take out last minute and mix without vortexing, spin down)
- Mix Master Mix well and gently (finger flick) and spin down
Add 30 µL of Mastermix to each sample from cDNA synthesis.
Mix well (pipetting) and spin down gently.
Run samples on pre-heated thermocycler with the program:
| A | B | C | |
| 95°C | 1 min | ||
| 98°C | 10 sec | repeat step 2, 18 times | |
| 65°C | 30 sec | ||
| 68°C | 3 min | ||
| 72°C | 10 min | ||
| 4°C | forever |
Note
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.
| Species | cDNA kit | Number of cycles | |
| Strombidium basimorphum | SMARTseq-v4 | 18 | |
| Prymnesium parvum | NEBNext | 25 | |
| Acantharia | SMARTseq-v4 | 18 | |
| Acantharia | NEBNext | 16 |
STOPPING POINT 4°C overnight
cDNA cleanup/bead purification
cDNA cleanup/bead purification
43m
43m
Preparations:
- Label for each sample two tubes PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Takara Bio Inc.Catalog #AM12230 . 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 (AMPure XP BeadsTakara Bio Inc. ), this needs to be very well and evenly mixed
- Aliquot beads, 22.5 µL x samples (plus extra)
- Bring the bead aliquot to Room temperature for at least 00:30:00
- Vortex the bead aliquot until evenly mixed
- Prepare fresh 80% EtOH, 400 µL x samples
30m
Add 22.5 µL of beads to each sample (amplified cDNA from the previous section)
Mix by pipetting up and down at least 10 times, and vortex
Incubate at Room temperature 00:08:00 to let cDNA bind to the beads
8m
Briefly spin down and place the samples on a Magnetic Stand-96Takara Bio Inc.Catalog #AM10027 for 00:05:00 or longer. Until the liquid appears completely clear and there are no beads in the supernatant.
Expected result
Not yet clear, beads have not yet all pelleted
clear, all beads have pelleted
5m
Pipet and discard the supernatant (72.5 µL), keeping the samples in the magnetic device
Keeping the samples in the magnetic device, add 200 µL fresh 80% EthanolTakara Bio Inc. to each sample.
Note
Do not disturb the beads
Wait 00:00:30
30s
Pipet and discard supernatant containing contaminants (use 100 µL)
Repeat the EtOH washing step for a total of 2 washing steps go to step #27
Briefly spin the samples to collect liquid off the sides
Place samples back in the magnetic device for00:00:30 , beads will again be collected on the side
30s
Remove all remaining ethanol/supernatant with a pipet (use 10 µL pipet)
Place samples at Room temperature for 00:02:00 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.
2m
Once the beads are dry add 15 µL of Elution buffer to all samples to cover the bead pellet
Remove samples from the magnetic device
Mix to re-suspend the beads by (multi)pipetting (can scrap of beads from the side)
Incubate at Room temperature for00:02:00 (longer) to rehydrate
2m
Briefly spin the samples to collect liquid off the sides
Place the samples back in the magnetic device for 00:01:00 , until the solution is completely clear
1m
Transfer the clear supernatant containing purified cDNA to PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Takara Bio Inc.Catalog #AM12230 tube (use 10 µL pipet).
Note
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
Make immediately an aliquot for Bioanalyzer analysis to prevent unnecessary freeze-thawing cycles.
STOPPING POINT - Label and store at -20 °C
cDNA Sample verification
cDNA Sample verification
Check the quality of cDNA by Agilent High Sensitivity DNA Kit Takara Bio Inc.Catalog #5067-4626 following the manufacture's protocol.
Expected result
Example of a desirable electrograph. Showing a good cDNA curve, and few primer dimers. Example of an acantharian sample. The peaks on the outsides are markers of the Bioanalyser chip for size and concentration marker
Quantify and calculate the concentration of cDNA. This is needed for the next cDNA library procedure.
cDNA library preparation and indexing – Nextera XT
cDNA library preparation and indexing – Nextera XT
Proceed with cDNA library preparation only for good quality samples from the previous step.
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.
Prepare to work very timely for this protocol
- Preheat a PCR thermocycler to 55 °C , with preheat lid at 100 °C
- Prepare from the Nextera XT DNA Library Preparation KitTakara Bio Inc.Catalog #FC-131-1096 the ATM and NT reagents in sufficient quantity (i.e. 5 ul per sample for each) separated over multiple tubes to facilitate multi-pipetting
Follow the Nextera XT DNA Library Preparation KitTakara Bio Inc.Catalog #FC-131-1096 manufacturer's protocol for " Tagment genomic DNA", and "Amplify Libraries", with the changes listed below.
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.
Store samples at 4 °C for up to 2 days or proceed immediately with purification
cDNA library purification
cDNA library purification
46m
46m
Preparations:
- Vortex the bead stock well (AMPure XP BeadsTakara Bio Inc. ), this needs to be very well and evenly mixed
- Aliquot beads, 30 µL x samples (plus extra)
- Bring the bead aliquot to Room temperature for at least 00:30:00
- Vortex the bead aliquot until evenly mixed
- Prepare fresh 80% EtOH, 400 µL x #samples
30m
Spin down your indexed cDNA samples (total 50 µL)
Add 30 µL of AMPure XP BeadsTakara Bio Inc. to each sample
- Mix by pipetting up and down
- Shake/vortex for00:02:00
2m
Incubate at Room temperature 00:05:00 to let cDNA bind to the beads
5m
Briefly spin down and place the samples on a Magnetic Stand-96Takara Bio Inc.Catalog #AM10027 for 00:05:00 or longer. Until the liquid appears completely clear and there are no beads in the supernatant.
Pipet and discard the supernatant (80 µL), keeping the samples in the magnetic device
Keeping the samples in the magnetic device, add 200 µL fresh 80% EthanolTakara Bio Inc. to each sample.
Note
Do not disturb the beads
Wait 00:00:30
Pipet and discard supernatant containing contaminants (use 100 µL pipet)
Repeat the EtOH washing step for a total of 2 washing steps go to step #52
Briefly spin the samples to collect liquid off the sides
Place samples back in the magnetic device for00:00:30 , beads will again be collected on the side
Remove all remaining ethanol/supernatant with a pipet (use 10 µL pipet)
Place samples at Room temperature for 00:05:00 minutes.
Until the pellet is no longer shiny, but before a crack appears. It needs to be ‘just’ dry, matte with no shine.
5m
Once the beads are dry add 52.5 µL of Resuspension BufferTakara Bio Inc. (NexteraXT kit) to all samples to cover the bead pellet
Remove samples from the magnetic device
Mix to re-suspend the beads by (multi)pipetting (can scrap of beads from the side)
Vortex for 00:02:00 followed by a very short spindown
2m
Incubate at Room temperature for00:02:00 to rehydrate
Briefly spin the samples to collect liquid off the sides
Place the samples back in the magnetic device for 00:02:00 , until the solution is completely clear
2m
Transfer the clear supernatant (50 uL) containing your purified cDNA library to PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Takara Bio Inc.Catalog #AM12230 tube (use 10 µL pipet).
Note
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
Make immediately an aliquot for Bioanalyser analysis to prevent unnecessary freeze-thawing cycles.
STOPPING POINT - Label and store at -20 °C for sequencing
cDNA library verification
cDNA library verification
Check the quality of the cDNA libraries by Agilent High Sensitivity DNA Kit Takara Bio Inc.Catalog #5067-4626 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.
Expected result
Example electrograph for a good cDNA library run with a Bioanalyser High Sensitivity DNA Kit (Agilent #5067-4626). Though primer-dimers are here still present and a follow up (repeat) cleaning is recommended. The region for smear analysis is indicated between blue lines. Example of an acantharian sample. The peaks on the outsides are markers.
Example electrograph for a desirable cDNA library run with a Bioanalyser High Sensitivity DNA Kit (Agilent #5067-4626). Example of an acantharian sample. The peaks on the outsides are markers.
Example electrograph for a desirable cDNA library run with a Bioanalyser DNA 7500 Kit (Agilent #5067-1506) instead of a Bioanalyser High Sensitivity DNA Kit (Agilent #5067-4626). This still allows for smear analysis though the concave parabola is less clear. This is more cost-effective than using a high sensitivity kit. Example of an acantharian sample The peaks on the outsides are markers.
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
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.