This protocol uses the Takara/Clontech SMART-seq HT Plus kit for carrying out RNA-seq when you have very low numbers of cells for input (10’s, 100’s or 1000’s of cells).
- Before starting
- Documentation
- What you’ll need
- A few important comments
- Sample preparation
- OPTION A: sorting into media
- OPTION B: sorting into lysis buffer
- Protocol
- Step 1: Generate cDNA from total RNA
- RNA extraction
- cDNA Synthesis
- Step 2: cDNA clean-up
- HT PLUS Library Prep
- A few important comments before you start
- Step 3: Adaptors
- Step 2: Amplification
- Step 3: Library Cleanup
- Quality Checks
- Sequencing Guidelines
- Normalize and Pool
Before starting
Documentation
Clontech ‘SMART-Seq HT PLUS’ for High-throughput single-cell mRNA-seq. This is an excellent kit for preparing cDNA very low amounts of RNA (as little as 1-100 cells). This kit is analagous to the well-known Clontech SMART-v4 kit, but is a faster/abbreviated workflow which combines RT/PCR steps and is roughly 30% lower cost compared to SMART-V4. The PLUS version includes both cDNA generation and library preparation all in one kit.
The protocol below uses the SMART-Seq HT kit, however another option for cDNA generation when starting with low-input is the Clontech Pico v2 kit. This has a few important difference from the SMART-Seq HT kit above. First, the Pico v2 kit uses random hexamer priming, rather than polyT, to make cDNA. This means that it works very well when your starting material is highly degraded (e.g. fixed cells or tissues). The random priming also means that this kit amplifies cDNA from all transcripts, including highly abundant RNA molecules such as rRNAs. We tend to use this kit if 1) investigators are working with highly degraded starting material, particularly FFPE tissues, or 2) if they really prefer to study total transcriptomes, rather than mRNAs.
What you’ll need
- 10x Lysis buffer
- RNase inhibitor (40U/ul)
- SeqAmp DNA Polymerase
- One-step Buffer
- SMART-Seq HT Oligonucleotide
- 3’ SMART-Seq CDS Primer II A
- SMARTScribe Reverse Transcriptase (100 U/µl)
- Nuclease-Free Water
- Elution Buffer (10 mM Tris-Cl, pH 8.5)
- Eppendorf LoBind DNA tubes
A few important comments
- Prior to sorting, stain cells in 10% complete media (RPMI with hepes, 10% FCS, Pen/Strep, NEAA, L-glut, Na-Pyruvate, 2ME, etc.)
- You’ll split this protocol into two days: day 1 will be your sort and cell lysis, day 2 will be RNA isolation and/or cDNA generation, and a modified NexteraXT library prep.
- Use only filter pipette tips and clean your area so it is free of RNases.
- Certain steps, such as 1st-strand cDNA synthesis must be carried out in a PCR clean hood
- Be sure to include the kit positive control and a water-only negative control with each experiment.
- the SMART-Seq HT kit uses a 96-well plate for mixing reactions. Do not use tubes.
- Similar to other Clontech low-input kits, SMART-Seq HT uses a template switching method to produce abundant cDNA directly from as few as 1-100 cells or from 10pg-1ng of total RNA.
- This protocol is very sensitive to variations in volume and other factors. Please make sure the pipettes are calibrated and avoid contamination.
Sample preparation
OPTION A: sorting into media
- Always use the 100 uM nozzle on the sorter (rather than 70 uM) and sort at low speed (~5-7K events/sec). This will be more gentle on the cells
- Sort into Lo-bind 96-well plates to prevent cells from sticking to sides (Lo-bind gives tighter pellets after spin down)
- Sort into complete media with 50% serum (RPMI with hepes, Pen/Strep, NEAA, L-glut, Na-Pyruvate, 2ME, etc.)
- Keep everything cold: cells, sort chamber, collection block, collection tubes, etc.
- Minimize time after sort to lysis. If sorting many samples, spin down in batches – don’t wait until the end if possible.
- Spin down cells. ~10,000 cells usually produce a visible pellet. Use a pipette tip to carefully draw off supe.
- lyse cells by adding 300 ul of buffer RLT (from Qiagen RNeasy kit) with 2-ME added fresh. Vortex to lyse cells.
- Flash freeze tube on dry ice and transfer to a prechilled box at -80°C.
OPTION B: sorting into lysis buffer
- Prepare 10x reaction buffer from the SMART-Seq HT kit by mixing 19 uL of 10x Lysis Buffer with 1 uL of RNase inhibitor. This is enough for ~20 samples. Scale up as needed, but be sure to maintain 19:1 ratio of lysis buffer to RNase inhibitor
- Prepare 1x reaction buffer by mixing 9.5 uL nuclease-free water with 1 uL of 10x reaction buffer. This is enough to sort one sample, so be sure to scale up as needed.
- Add 5ul of 1x reaction buffer to collection eppendorf tube (or one well of a 96-well plate) and sort directly into this tube or well.
- Immediately after sample is sorted, add an additional 5.5 uL of 1x reaction buffer to tube.
- Store at -80°C until ready to begin cDNA synthesis. You can proceed directly from this cell lysate to cDNA preparation with no intermediate RNA isolation step.
Protocol
Step 1: Generate cDNA from total RNA
RNA extraction
- If you chose Option A above, extract RNA using the the Qiagen RNeasy micro kit and store at -80°C.
- If you chose Option B above, skip RNA extraction and proceed directly to cDNA synthesis
- Assess RNA integrity using an Agilent Tapestation 4200 and High-sensitivity RNA screentape. Although tapestation is not ideal for estimating concentration of RNA, it can be used in this case as a rough estimate, since you may not have enough material to quantify using Qubit.
- If you have sample to spare, you can try to measure RNA concentration using Qubit, but it is unlikely to be above the limit of sensitivity (2 ng/ul). A more practical approach is the use the concentrations provided by High-sensitivity RNA tapestation run. These aren’t ideal, but they’ll have to do.
- Especially if working with unfamiliar samples, including positive and negative controls is encouraged. Negative controls can be 10.5 of Nuclease-free water. Positive controls must be included in the cDNA step and products can move forward with dilution just like experimental samples.
cDNA Synthesis
Name | Location | Action |
---|---|---|
One-step Buffer | -20°C freezer | Thaw at room temperature |
10X Reaction Buffer | -20°C Freezer | If made, thaw on ice; if not made, see below |
Nuclease-free Water | -20°C freezer | Thaw at room temperature |
SMART-Seq HT Oligonucleotide | -20°C freezer | Thaw on ice |
RNase Inhibitor (RRI) | -20°C freezer | Thaw on ice |
Elution Buffer | -20°C freezer | Thaw at room temperature |
SeqAmp DNA Polymerase | -20°C freezer | Store until needed |
SMARTScribe Reverse Transcriptase | -20°C freezer | Store until needed |
Kit reagent | Volume per rxn (uL) |
---|---|
Nuclease-free water | 0.77 |
One-Step buffer | 8.8 |
SMART-Seq HT Oligonucleotide | 1.1 |
RNase Inhibitor | 0.55 |
Kit reagent | Volume per rxn (uL) |
---|---|
SeqAmp DNA Polymerase | 0.33 |
SMARTScribe Reverse Transcriptase | 2.2 |
Input of total RNA (or amount of cells) | Typical number of PCR cycles |
---|---|
1 ng (or about 100 cells) | 14-15 |
100 pg (or about 10 cells) | 17-18 |
10 pg (or about 1 cells) | 18 |
Step 2: cDNA clean-up
HT PLUS Library Prep
A few important comments before you start
- You'll need 8uL of cDNA, diluted to a concentration of 0.125 ng/ul - 1.25 ng/ul or a total input of 1 - 10ng. All samples should have the same input.
- If your sample is less than 0.125 ng/ul, you can attempt to move forward with the library prep without dilution. cDNA for negative controls of this kit can have up to 100pg/ul of contamination after 17 PCR cycles. If you have a low concentration sample, judge based on your Qubit concentration and cycles used whether to move forward. Tapestation concentrations are typically inaccurate for this.
Step 3: Adaptors
Name | Location | Action |
---|---|---|
Stem-loop adaptors | -20°C freezer, purple cap | Thaw on ice |
FE Dilution Buffer | -20°C freezer, white cap | Thaw on ice |
Lib Prep Buffer | -20°C freezer, blue cap | Thaw on ice |
Rxn Enhancer | -20°C freezer, red cap | Thaw on ice |
Amp Buffer | -20°C freezer, orange cap | Thaw on ice |
10X FE | -20°C freezer, white cap | Store until needed |
Lib Prep Enzyme | -20°C freezer, blue cap | Store until needed |
PrimeSTAR DNA Polymerase | -20°C freezer, green cap | Store until needed |
Nuclease-free water | -20°C freezer | Thaw at room temperature |
Unique Dual Indexes | -20°C freezer | Thaw at room temperature |
Name | Volume |
---|---|
Library Prep Buffer | 4.4 |
Rxn Enhancer | 3.85 |
Library Prep Enzyme | 2.2 |
1X FE | 1.1 |
Step 2: Amplification
Name | Volume |
---|---|
Amplification Buffer | 23.65 ul |
PrimeSTAR HS DNA Polymerase | 1.1 ul |
Step 3: Library Cleanup
Quality Checks
Perform quality control steps :
Libraries can be stored at -20°C for up to 30 days.