Single cell RNA-seq is a pretty popular topic right now. Most of the kits are really expensive and if you are new to preparing sequencing libraries, you are not likely to be successful in the first couple tries.
This protocol uses the SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing by Takara, compare to the Takara user manual, this protocol reduces cost of reagent by half, with a successful library construction rate near 100%.
Like many of its kits, the Takara SMART-Seq takes advantage of some run-off effects of the Moloney Murine Leukemia Virus (MMLV) derived reverse transcriptase and adds an oligonucleotide to the 5’ end of the RNA at the end of the cDNA synthesis for each RNA molecule. This is called “Template Switching”.
In the future, I will do some other single cell RNA-seq protocols, such as Drop-seq.
Other than standard lab supplies, you will need the following reagents:
-SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing by Takara
(we will be able to do 24 reactions with a 12 reactions kit J)
– AMPure XP Beads
– ERCC RNA Spike-In Mix (optional, but I highly recommend it)
– Qubit dsDNA HS Assay Kit
– Qubit Fluorometer
– Qubit assay tubes
– Agilent Bioanalyzer High Sensitivity DNA Analysis Kits
– Nextera XT DNA Library Preparation Kit
- First, sort individual cells into a 96-well plate, or 0.2ml PCR tubes, using a method of your choice. e.g. FACS. The liquid volume should be under 3.75µl.
- Keep samples and reagents on ice for as long as you can.
- Vortex buffer and primers and spin down briefly before use!
Prepare a 10X lysis buffer:
19µl 10X lysis buffer
1µl RNase inhibitor
20µl total
- Mix by pipetting
- Make a fresh 4million fold dilution of ERCC spike in from stock.
Cell lysis:
0.5-3.75µl cell
Xµl Nuclease Free water (3.75µl – vol. of cell)
1µl ERCC spike in dilution
0.5µl 10X lysis buffer (Make a master mix with nuclease-free water, 0.5µl alone is difficult to pipette!)
5.25µl total
- Incubate room temperature 5min, then 10min on ice.
Reverse Transcription:
- First, make 1/100 dilution of 3’ SMART-Seq CDS Primer II A (12µM)
5.25µl cell lysed
0.5µl Nuclease-free water
0.5µl 3’ SMART-Seq CDS Primer II A (1/100 dilution)
72˚C 3min, transfer immediately on ice, incubate for additional 2min
Make RT Master Mix:
2µl 5X Ultra Low First-Strand Buffer
0.5µl SMART-Seq V4 Oligonucleotide (48µM)
0.25µl RNase inhibitor
1µl SMARTScribe Reverse Transcriptase
3.75µl total
- Add RT Master Mix to the cell lysis + CDS primer. The total volume of the RT reaction is 10µl. Incubate 42˚C 90min, 70˚C 10min, 4˚C hold.
Safe Stopping Point: the RNA are now transcribed to cDNA, which is more stable, the samples could be stored in -20˚C for weeks.
Pre-amplification PCR:
- First, make a 1/200 dilution of PCR_ Primer II A
12.5µl 2X SeqAmp PCR Buffer
1µl PCR Primer II A dilution
0.5µl SeqAmp DNA Polymerase
1.5µl nuclease free water
10µl RT reaction
25µl total
- Make a master mix of 2X SeqAmp PCR Buffer, PCR Primer II A dilution, SeqAmp DNA Polymerase, and water, mix well be pipetting, then add to the RT reactions
- 95˚C 1min
- 98˚C 10s
- 65˚C 30s
- 68˚C 3min
- go back to step 2 for 19 more cycles, so 20 cycles total
- 72˚C 10min
4˚C hold
*The number of cycles will depend on the cell type. But 20 should be a good start.
- (Optional) Quantify dsDNA in reactions using Qubit dsDNA HS kit, the DNA concentration should be between 2-3 ng/µl. If the concentration is low, place the reaction back on the thermal cycler, add 3 cycles, and measure again.
- Purify PCR reaction with 0.8X AMPure XP Beads
- Quantify purified cDNA with Qubit DNA HS assay, dilute to ~500pg-1ng/µl, run on a Bioanalyzer DNA HS chip.
Bad: degraded RNA Good: full length RNA
Safe stopping point: The purified cDNA can be stored in -20˚C for months.
Sequencing Library Construction with Nextera XT kit
With our double-stranded, pre-amplified cDNA, we can now use the Nextera XT kit to make dual indexed libraries so up to 96 single cell libraries could be pooled and sequenced together.
The nextera kit makes use of some special properties of the viral transposons and contains a synthetic version which can cut dsDNA and ligate adaptors at cutting sites at the same time. During the tagmentation step, the full-length ds cDNA are cut to approximately 300 bp and PCR handles are added to the ends of the cutting sites. During the PCR step, longer, indexed primers which part of them matches the previous added-on PCR handles are used to amplify sequencing libraries. These longer primers also contain reads that are compatible with illumina sequencers.
After this step, our library construction is finished and we will move to clean up and QC before finally sending them off for sequencing.
Of course, as penny pinchers, we will only use ¼ of reaction amounts recommended by illumina for each sample, so a 24rxn kit could be used for 96 samples.
- Dilute cDNA sample to ~160pg/µl, it is important that the total input of cDNA is ~200pg
Tagmentation:
- make a master mix of TD buffer and Amplicon Tagment Mix(ATM)
2.5µl TD Buffer
1.25µl ATM
3.75µl
+ 1.25µl cDNA dilution
5µl total
Incubate on a thermal cycler 55˚C 10min, keep at 10˚C
- While the reaction is kept at 10˚C, add 1.25µl NT buffer and mix well incubate at room temperature for 5mins.
PCR:
- add the following mix to neutralized tagmentation reaction
3.75µl NPM
1.25µl primer N70X
1.25µl primer N50X
12.5µl total
Stage1: 72˚C 3min
95˚C 30s
Stage2: 95˚C 10s
55˚C 30s
72˚C 30s
Cycle stage2 for 12 cycles
Stage3: 72˚C 5min, 4˚C hold
-Take 2µl from each single cell library and pool them together. Perform purification with either ampure beads or pippin prep.
– The size-selected libraries could be checked by loading onto a Bio-analyzer DNA HS chip. Now your library is ready for sequencing on an illumina platform!
RNA-seq: Protocols that Work! 