This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
MATQ-seq protocol
Chenghang (Chuck) Zong
Baylor College of Medicine
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Quantification of transcriptional variations in single cells, particularly of the same cell population, is currently still limited by both the sensitivity and the technical noise of single-cell RNA-seq assays. We report a single-cell RNA-seq method -- Multiple Annealing and dC-Tailing based Quantitative single-cell RNA-seq (MATQ-seq) -- that achieves high sensitivity and low technical noise.
Keywords
Single cell RNA-seq; single cell transcriptome
- Cell lysis Lysis solution:
0.2% Triton-X (Sigma) in DEPC H2O 1μL
0.1mM DTT (Life Technologies) 0.05μL
RnaseOUT (Life Technologies) 0.05μL
Primer**** mix 0.4μL (PAGE purified from IDT; GAT27dT 1.5uM, GAT275N3G 5uM, GAT275N3T 5uM)
10Mm each dNTP 0.1μL
Pipette single cell in lysis solution, briefly centrifuge, then put on PCR block 72ºC 3min, then quickly put on ice for at least 1min.
Reverse transcription
Add RT mix containing:
5X RT buffer (Life Technologies) 0.8μL
0.1mM DTT 0.2μL
RnaseOUT 0.1μL
Superscript III (Life Technologies) 0.15μL
Rnase-free H2O 1.15μL
Mix well by pipetting with low retention tips
Run following program:
10 cycles of
8ºC 12s
15ºC 45s
20ºC 45s
30ºC 30s
42ºC 2min
50ºC 3min
End cycles
50ºC 15min
4 ºC Forever
- Digestion of primers 50ºC 1min
Add 0.2μL T4 DNA polymerase (New England Biolabs) at 37ºC, mix well
37ºC 40min
75ºC 20min
37ºC 40min
80ºC 20min
4 ºC Forever
- RNA digestion Add 0.1μL RnaseH (New England Biolabs), 0.1μL RnaseI (New England Biolabs), mix well
37ºC 15min
72ºC 15min
4 ºC Forever
- Tailing make following TdT mix:
10X TdT buffer (New England Biolabs) 0.4μL
100mM dCTP (Life Technologies) 0.4μL
TdT terminal transferase (New England Biolabs) 0.1μL
H2O 3.1μL
Add the TdT mix into sample, mix well
37ºC 15min
72ºC 15min
4 ºC Forever
- Second strand synthesis Make following 2nd stand mix:
10X Thermopol (New England Biolabs) 1.5μL
dNTP (10uM each) 1.25μL
10uM GAT21 6n3G pimer**** 1.25μL
H2O 12μL
Add the mix into sample, heat the block to
95ºC 1min
48ºC hold, add Deepvent exo- DNA polymerase (New England Biolabs) 0.4μL, mix well
10 cycles of
48ºC 20s
72ºC 1min
End cycles
72ºC 2min
4 ºC Forever
Amplification
10X Thermopol Buffer 13μL
100uM GAT27 primer**** 0.8μL
10mM each dNTP 3μL
PCR grade water 116μL
Deepvent exo- DNA polymerase 3μL
Split into 4 PCR tubes
95ºC 30s
24 cycles of
95 ºC 15s
62 ºC 20s
72 ºC 2min
End cycles
72 ºC 5min
4 ºC Forever
Use Qiagen PCR purification kit to purify the end product
3NGAT24 Double strand conversion (For better cluster identification during sequencing)
200ng of amplified cDNA
10X Thermopol buffer 5μL
dNTP 1.25μL
100Mm 3NGAT24 primer**** 0.25μL
Deepvent exo- DNA polymerase 1μL
Add H2O to total 50 μL
95ºC 30s
20 cycles of
62 ºC 20s
72 ºC 1 min
End cycles
72 ºC 3min
95ºC 30s
20 cycles of
62 ºC 20s
72 ºC 1 min
End cycles
72 ºC 3min
4 ºC Forever
Use 1.2X Ampure XP beads (60 μL) to purify
**** Primer sequence (Integrated DNA Technologies, PAGE purified):
GAT27dT: GTG AGT GAT GGT TGA GGA TGT GTG GAG NNNNN TTTTTTTTTTTTTTTTTTTT
GAT27 5N3G: GTG AGT GAT GGT TGA GGA TGT GTG GAG NNNNN GGG
GAT27 5N3T: GTG AGT GAT GGT TGA GGA TGT GTG GAG NNNNN TTT
GAT21 6N3G: GAT GGT TGA GGA TGT GTG GAG NNNNNN GGG
GAT27 PCR: GTG AGT GAT GGT TGA GGA TGT GTG GAG
3NGAT24: NNN AGT GAT GGT TGA GGA TGT GTG GAG
This is a list of supplementary files associated with this preprint. Click to download.
- MATQseqsinglecelltranscriptomeprotocolNE.docx
MATQ seq single cell transcriptome protocol Zong Lab
Version History
CURRENT STATUS: Posted
Version 1
Posted 28 Feb, 2017
Metrics
Comments: 0
HTML Views: ...
Associated Publications
Effective detection of variation in single-cell transcriptomes using MATQ-seq
by Kuanwei Sheng, Wenjian Cao, Yichi Niu, +2
Nature Methods (28 February, 2017)
Method Article
MATQ-seq protocol
Chenghang (Chuck) Zong
Baylor College of Medicine
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 28 Feb, 2017
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Quantification of transcriptional variations in single cells, particularly of the same cell population, is currently still limited by both the sensitivity and the technical noise of single-cell RNA-seq assays. We report a single-cell RNA-seq method -- Multiple Annealing and dC-Tailing based Quantitative single-cell RNA-seq (MATQ-seq) -- that achieves high sensitivity and low technical noise.
- Cell lysis Lysis solution:
0.2% Triton-X (Sigma) in DEPC H2O 1μL
0.1mM DTT (Life Technologies) 0.05μL
RnaseOUT (Life Technologies) 0.05μL
Primer**** mix 0.4μL (PAGE purified from IDT; GAT27dT 1.5uM, GAT275N3G 5uM, GAT275N3T 5uM)
10Mm each dNTP 0.1μL
Pipette single cell in lysis solution, briefly centrifuge, then put on PCR block 72ºC 3min, then quickly put on ice for at least 1min.
Reverse transcription
Add RT mix containing:
5X RT buffer (Life Technologies) 0.8μL
0.1mM DTT 0.2μL
RnaseOUT 0.1μL
Superscript III (Life Technologies) 0.15μL
Rnase-free H2O 1.15μL
Mix well by pipetting with low retention tips
Run following program:
10 cycles of
8ºC 12s
15ºC 45s
20ºC 45s
30ºC 30s
42ºC 2min
50ºC 3min
End cycles
50ºC 15min
4 ºC Forever
- Digestion of primers 50ºC 1min
Add 0.2μL T4 DNA polymerase (New England Biolabs) at 37ºC, mix well
37ºC 40min
75ºC 20min
37ºC 40min
80ºC 20min
4 ºC Forever
- RNA digestion Add 0.1μL RnaseH (New England Biolabs), 0.1μL RnaseI (New England Biolabs), mix well
37ºC 15min
72ºC 15min
4 ºC Forever
- Tailing make following TdT mix:
10X TdT buffer (New England Biolabs) 0.4μL
100mM dCTP (Life Technologies) 0.4μL
TdT terminal transferase (New England Biolabs) 0.1μL
H2O 3.1μL
Add the TdT mix into sample, mix well
37ºC 15min
72ºC 15min
4 ºC Forever
- Second strand synthesis Make following 2nd stand mix:
10X Thermopol (New England Biolabs) 1.5μL
dNTP (10uM each) 1.25μL
10uM GAT21 6n3G pimer**** 1.25μL
H2O 12μL
Add the mix into sample, heat the block to
95ºC 1min
48ºC hold, add Deepvent exo- DNA polymerase (New England Biolabs) 0.4μL, mix well
10 cycles of
48ºC 20s
72ºC 1min
End cycles
72ºC 2min
4 ºC Forever
Amplification
10X Thermopol Buffer 13μL
100uM GAT27 primer**** 0.8μL
10mM each dNTP 3μL
PCR grade water 116μL
Deepvent exo- DNA polymerase 3μL
Split into 4 PCR tubes
95ºC 30s
24 cycles of
95 ºC 15s
62 ºC 20s
72 ºC 2min
End cycles
72 ºC 5min
4 ºC Forever
Use Qiagen PCR purification kit to purify the end product
3NGAT24 Double strand conversion (For better cluster identification during sequencing)
200ng of amplified cDNA
10X Thermopol buffer 5μL
dNTP 1.25μL
100Mm 3NGAT24 primer**** 0.25μL
Deepvent exo- DNA polymerase 1μL
Add H2O to total 50 μL
95ºC 30s
20 cycles of
62 ºC 20s
72 ºC 1 min
End cycles
72 ºC 3min
95ºC 30s
20 cycles of
62 ºC 20s
72 ºC 1 min
End cycles
72 ºC 3min
4 ºC Forever
Use 1.2X Ampure XP beads (60 μL) to purify
**** Primer sequence (Integrated DNA Technologies, PAGE purified):
GAT27dT: GTG AGT GAT GGT TGA GGA TGT GTG GAG NNNNN TTTTTTTTTTTTTTTTTTTT
GAT27 5N3G: GTG AGT GAT GGT TGA GGA TGT GTG GAG NNNNN GGG
GAT27 5N3T: GTG AGT GAT GGT TGA GGA TGT GTG GAG NNNNN TTT
GAT21 6N3G: GAT GGT TGA GGA TGT GTG GAG NNNNNN GGG
GAT27 PCR: GTG AGT GAT GGT TGA GGA TGT GTG GAG
3NGAT24: NNN AGT GAT GGT TGA GGA TGT GTG GAG
Associated Publications
Effective detection of variation in single-cell transcriptomes using MATQ-seq
by Kuanwei Sheng, Wenjian Cao, Yichi Niu, +2
Nature Methods (28 February, 2017)