Dec 02, 2021
- cecilia 1,
- Suzie Alarcon1,
- Alessandro Sette1
- 1La Jolla Institute for Immunology
Protocol Citation: cecilia , Suzie Alarcon, Alessandro Sette 2021. SMART-Seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bwu5pey6
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
Created: July 24, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 51837
Keywords: Cell lysis, Oligot-dT priming, Reverse transcription, PCR preamplification, Quality Check cDNA, Tagmentation reaction, ASAPCRN, reverse transcription, seq this protocol, cell lysi, seq, pcr preamplification, cell
Abstract
This protocol details the cell lysis / Oligot-dT priming, reverse transcription, PCR preamplification and quality Check cDNA and tagmentation reaction.
Attachments
d2bxbg4xf.docx
393KB
Materials
REAGENTS:
- PBS (phosphate buffered saline) with no Ca/MgThermo Fisher ScientificCatalog #Invitrogen 14190-144
- TrypLE™ Express Enzyme (1X), no phenol redThermo FisherCatalog #12604021
- RNaseZapAmbionCatalog #AM9780
- DNA-OFF™Takara Bio Inc.Catalog #9036
- Triton™ X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787
- dNTP Mix (dATP, dCTP, dGTP, and dTTP, each at 10mM) Thermo Fisher ScientificCatalog #R0192
- First-strand buffer (5×; 250 mM Tris-HCl, pH 8.3, at room temperature (25 °C); 375 mM KCl; 15 mM MgCl2; Invitrogen, cat. no. 18064-014)
- Superscript IIInvitrogen - Thermo FisherCatalog #18064-014
- DTT (Invitrogen, cat. no. 18064-014)
- Recombinant RNAse InhibitorTakara Bio Inc.Catalog #2313A
- Betaine BioUltra ≥99.0% (NT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #61962
- 1 M Magnesium Chloride (MgCl2)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M8266
- Kapa HiFi Hotstart ReadymixKapa BiosystemsCatalog #KK2601
Critical: A HotStart DNA polymerase is necessary to minimize the background amplification when working with single cells and is more practical when working with automated liquid-handling platforms.
- Agencourt AMPure XP beadsBeckman CoulterCatalog #A63881
- Ethanol 99.5% (vol/vol); Kemethyl, cat. no. SN366915-06)
Caution: It is flammable; handle it using appropriate safety equipment.
- Elution Buffer (EB)QiagenCatalog #19086
- TruSeq Dual Index Sequencing Primer BoxIllumina, Inc.Catalog #FC-121-1003
- Nextera XT DNA Sample Preparation Kit, 96 samplesIllumina, Inc.Catalog #FC-131-1096
- Nextera XT Index Kit (24 indexes 96 samples)Illumina, Inc.Catalog #FC-131-1001
Troubleshooting
Cell Lysis / Oligot-dT Priming
6m 20s
Note
Timing: ∼15 min (for eight-strip tubes)
Dilute the oligo-dT30VN primer to 10 micromolar (µM) by adding 10 µL of 100 micromolar (µM) oligo-dT primers and 90 µL of nuclease-free water to a tube and mix well.
Prepare cell lysis buffer by adding 1 µL of RNase inhibitor to 19 µL of a 0.2% (vol/vol) Triton X-100 solution.
Note
If you are working with purified RNA, this step can be omitted and a corresponding volume of water can be used instead.
Isolate single cells in the lowest possible volume (preferably ≤0.5 µL , possibly 0.3 µL ) or pipet the appropriate amount of RNA into a 0.2 mL thin-walled PCR tube. Single cells can be obtained either by using a micro capillary pipette or via FACS.
Place each single cell into a 0.2 mL thin-walled PCR tube containing 2 µL of cell lysis buffer, 1 µL of oligo-dT primer and 1 µL of dNTP mix.
Quickly vortex the tube to mix, and then spin down the solution (700 g for 00:00:10 at Room temperature ) and immediately place it On ice .
10s
Incubate the samples at 72 °C for 00:03:00 and immediately put the tube back On ice .
3m
Spin down the samples (700 g for 00:00:10 at Room temperature ) to collect the liquid at the bottom of the tubes, and then put them immediately back On ice .
Note
The oligo-dT primer is now hybridized to the poly(A) tail of all the mRNA molecules.
Purified RNA:
Xul RNA up to 2.5ul
1ul oligo-dT Primer (10uM)
1ul dNTP (10mM)
xul H2O
4.5ul Total
72 °C for 00:03:00 , snap cool.
3m 10s
Reverse Transcription
Prepare the RT mix for all reactions plus one additional reaction by combining and mixing the reagents listed in the table below.
| A | B | C | |
| Component | Volume (ul) | Final Conc | |
| Superscipt II | 0.50 | 100 U | |
| RNAse inhibitor (40 U/ul) | 0.25 | 10 U | |
| Superscript II FS buffer (5X) | 2.00 | 1X | |
| DTT (100 mM) | 0.50 | 5 mM | |
| Betaine (5 M) | 2.00 | 1 M | |
| MgCl2 (1 M) | 0.06 | 6 mM | |
| TSO (100uM) | 0.10 | 1 uM | |
| H2O | 0.29 | - | |
| Total | 5.70 | - |
Add 5.7 µL of RT mix to Samples for a total of 10 µL .
Spin and incubate as follows:
| A | B | C | D | |
| Cycle | Temperature (°C) | Time | Purpose | |
| 1 | 42 | 90 min | RT and template-switching | |
| 2–11 | 50 | 2 min | Unfolding of RNA secondary structures | |
| 42 | 2 min | Completion/continuation of RT and template-switching | ||
| 12 | 70 | 15 min | Enzyme inactivation | |
| 13 | 4 | Hold | Safe storage |
PCR preamplification
Prepare the PCR mix for all reactions plus one additional reaction by combining and mixing the following components:
| A | B | C | |
| Component | Volume (μl) | Final concentration | |
| First-strand reaction | 10 | – | |
| KAPA HiFi HotStart ReadyMix (2×) | 12.50 | 1× | |
| IS PCR primers (10 μM) | 0.25 | 0.1 μM | |
| Nuclease-free water | 2.25 | – | |
| Total volume | 25 | – |
Add 15 µL of PCR mix to each tube from Step 12, which contains the first-strand reaction and perform the PCR in a thermal cycler by using the following program:
| A | B | C | D | E | |
| Cycle | Denature | Anneal | Extend | Hold | |
| 1 | 98 °C, 3 min | – | – | – | |
| 2–19 (see below) | 98 °C, 20 s | 67 °C, 15 s | 72 °C, 6 min | – | |
| 20 | – | – | 72 °C, 5 min | – | |
| 21 | – | – | – | 4 °C |
| A | B | C | |
| Input Amount Total RNA | Input Amount, Cells | Typical No. of PCR Cycles | |
| 10 ng | 1,000 cells | 12 | |
| 1 ng | 100 cells | 12 | |
| 500 pg | 50 cells | 13 | |
| 100 pg | 10 cells | 15 | |
| 10 pg | 1 cell | 18 |
Ampure Cleanup
Perform a typical Ampure cleanup using 1:1 ratio of Ampure:cDNA.
Elute using 17.5 µL EB solution and pipette 15 µL to transfer to a new tube.
Quality Check cDNA
Run a High Sensitivity Bioanalyzer Chip to check for quality of cDNA.
A good library should be free of short (<500 bp) fragments and should show a peak at 1.5–2 kb.
Tagmentation Reaction
10m
Setup the tagmentation RXN as follows:
| A | B | C | |
| Component | Volume (μl) | Final concentration | |
| Tagment DNA buffer (TD, 2×) | 10 | 1× | |
| Amplicon tagment mix | 5 | – | |
| DNA from PCR | Variable | – | |
| Nuclease-free water | Variable | – | |
| Total volume | 20 | – |
Incubate in a thermal cycler at 55 °C for 00:05:00 and bring to 4 °C HOLD.
5m
Add 5 µL of NT buffer to the 20 µL RXN and mix.
Incubate at Room temperature for 00:05:00 .
5m
Enrichment of Tagmented cDNA
Prepare the following PCR RXN as follows:
| A | B | |
| Component | Volume (μl) | |
| DNA | 25 | |
| Nextera PCR master mix | 15 | |
| Index 1 primers (N7xx) | 5 | |
| Index 2 primers (N5xx) | 5 | |
| Total volume | 50 |
Run the PCR RXN on a thermal cycler with the following conditions:
| A | B | C | D | E | |
| Cycle | Denature | Anneal | Extend | Hold | |
| 1 | – | – | 72 °C, 3 min | – | |
| 2 | 95 °C, 30 s | – | – | – | |
| 3–14* | 95 °C, 10 s | 55 °C, 30 s | 72 °C, 30 s | – | |
| 15 | – | – | 72 °C, 5 min | – | |
| 16 | – | – | – | 4 °C |
*for 1ng, 8-12 cycles could be used
Expected results:
Note
Using Single Cell (thus, purified RNA should yield better stats)
Sequence reads from each individual cell are normally in the range of 1–20 million, depending on the level of multiplexing in the sequencing. When sequencing 50-bp single-end reads, we find that normally 60% of reads map uniquely to the genome (20% multimapping and 20% with no match); of the uniquely mapping reads, >60% of the reads map to annotated RefSeq exons, 20% intronic and 20% intergenic, but these values depend on the completeness of the gene annotations. The read coverage across transcripts should be even.