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
Barcoding lentiviral Cre vectors for use in experiments involving downstream Tuba-seq analysis.
Zoe Rogers
Winslow Lab
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
This protocol outlines the procedure to massively barcode Lentiviral Cre vectors. This procedure was developed for use of the Tuba-seq pipeline to calculate tumor sizes in lentivirally inducible mouse models of cancer. The protocol accompanies Rogers et al, Nature Methods, published online May 22, 2017 (nmeth.4297).
Keywords
Tuba-seq, barcoding, lentivirus, Cre
Vectors (All available at AddGene):
Vector Name
AddGene ID
Lenti-sgNT1/Cre
66895
Lenti-sgNT3/Cre
89654
Lenti-sgNeo1/Cre
67594
Lenti-sgNeo2/Cre
89652
Lenti-sgNeo3/Cre
89653
Lenti-sgSmad4/Cre
89651
Lenti-sgSetd2#1/Cre
89649
Lenti-sgSetd2#2/Cre
89650
Lenti-sgRbm10/Cre
89648
Lenti-sgRb1/Cre
89647
Lenti-sgp53/Cre
89646
Lenti-sgKeap1/Cre
89645
Lenti-sgCdkn2a/Cre
89644
Lenti-sgAtm/Cre
89643
Lenti-sgArid1a/Cre
89642
Lenti-sgApc/Cre
89641
Lenti-sgLkb1/Cre
66894
Oligos:
Universal Reverse Primer (5’ → 3’):
AGCTAGGGATCCGCCGCATAACCAGTG
Barcoded Forward Primer (5’ → 3’):
AGCTAGTCCGGXXXXXXXXAANNNNNTTNNNNNAANNNNNATGCCCAAGAAGAAGAGGAAGGTGTC
NOTE: 8-nucleotide ID specific to each individual sgRNA (sgID; X) and the 15-nucleotide random barcode (BC; N). sgID is optional, if you are not creating a pool of vectors, the second barcode may not be necessary for your experience.
Reagents:
- XmaI (NEB, R0180)
- BspEI (NEB, R0540)
- T4 DNA Ligase (NEB, M0202)
- PrimeSTAR® HS DNA Polymerase (premix) (Clontech, R040A)
- Qiagen® PCR Purification Kit (Qiagen, 28106)
- ElectroMAX DH10B cells (Thermo Fisher, 18290015)
- 0.1 cm GenePulser/MicroPulser Cuvettes (Bio-Rad, 165-2089)
- Qiagen® HiSpeed MidiPrep Kit (Qiagen, 12643)
- Qiagen® MinElute Gel Extraction Kit (Qiagen, 28604)
BD MicroPulser™ Electroporator (Bio-Rad,165-2100)
PCR amplify the barcoded insert off of Lenti-PGK/Cre plasmid.
Reaction mix:
200 l PrimeStarHS Mix
12 l Forward Primer (10 M)
12 l Reverse Primer (10 M)
8 l Template (500 ng/L)
168 l H20
400 l Total Split into 4, 100 l reactions
PCR Program:
Temp. (°C) Time (min)
- 98 1:00
- 98 0:15
- 55 0:05
- 72 1:00
- Go to step 2, 34x
- 72 5:00
4 forever
- Pool the PCR reactions and PCR purify using the Qiagen® PCR Purification Kit. Elute in 35 l H20.
- Digest barcoded insert with BspEI and BamHI. Reaction mix:
5 l Buffer 3.1
1.5 l BspEI
1.5 l BamHI
3 g Barcode Insert
H20 to 50 l
50 l Total Incubate at 37°C 2 hours
- Concurrently digest Lentiviral Plasmid with XmaI and BamHI. Reaction mix:
5 l Cutsmart Buffer
1.5 l XmaI
1.5 l BamHI
5 g Lentiviral Plasmid
H20 to 50 l
50 l Total Incubate at 37°C 2 hours
- Run out digested products on a 2% agarose gel and gel extract the fragments using the Qiagen® MinElute Gel Extraction Kit (Qiagen, 28604) and elute in 25 l of H20.
- Ligate digested barcoded insert into digested Lentiviral Plasmid Reaction mix:
8 l Ligation Buffer
1.6 l T4 DNA Ligase (NEB, M0202)
300 ng Digested Lentiviral Plasmid
180 ng Digested Barcoded Insert
H20 to 80 l
80 l Total Incubate at 16°C overnight
- PCR purify the ligation product using the Qiagen® PCR Purification Kit (Qiagen, 28106)
- Transform 1 l into 20 l ElectroMAX DH10B cells (Thermo Fisher, 18290015). Use 0.1 cm GenePulser/MicroPulser Cuvettes (Bio-Rad, 165-2089) and BD MicroPulser™ Electroporator (Bio-Rad,165-2100)at 1.9 kV.
- Ad 500 l of SOC media and shake at 200 rpm for 30 minutes at 37°C.
- Plate on LB-Amp plates (1x1 l, 1x 10 l, and 5x 100 l) and incubate at 37°C overnight.
- Count colonies on either the 1 l or 10 l plate and use these counts to estimate the colony number on the 100 l plates. The total number of colonies counted will provide an estimate of the number of unique barcodes obtained.
- Scrape bacterial colonies off of the LB-Amp plates in 10 mL of LB-Amp per plate.
- Pool liquid in a flask and shake at 200 rpm for 30 minutes at 37°C.
- Midi-prep the barcoded plasmid pool using the Qiagen® HiSpeed MidiPrep Kit (12643).
- Determine plasmid concentration and confirm barcoding by Sanger sequencing.
2-3 full days.
The transformation will be inhibited if there is any salt left in the transformed plasmid solution.
You may need to titrate the voltage on the electroporater, depending on the machine they behave differently.
Version History
CURRENT STATUS: Posted
Version 1
Posted 25 May, 2017
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Subject Areas
Molecular Biology
Associated Publications
Method Article
Barcoding lentiviral Cre vectors for use in experiments involving downstream Tuba-seq analysis.
Zoe Rogers
Winslow Lab
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 25 May, 2017
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Molecular Biology
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
This protocol outlines the procedure to massively barcode Lentiviral Cre vectors. This procedure was developed for use of the Tuba-seq pipeline to calculate tumor sizes in lentivirally inducible mouse models of cancer. The protocol accompanies Rogers et al, Nature Methods, published online May 22, 2017 (nmeth.4297).
Vectors (All available at AddGene):
Vector Name
AddGene ID
Lenti-sgNT1/Cre
66895
Lenti-sgNT3/Cre
89654
Lenti-sgNeo1/Cre
67594
Lenti-sgNeo2/Cre
89652
Lenti-sgNeo3/Cre
89653
Lenti-sgSmad4/Cre
89651
Lenti-sgSetd2#1/Cre
89649
Lenti-sgSetd2#2/Cre
89650
Lenti-sgRbm10/Cre
89648
Lenti-sgRb1/Cre
89647
Lenti-sgp53/Cre
89646
Lenti-sgKeap1/Cre
89645
Lenti-sgCdkn2a/Cre
89644
Lenti-sgAtm/Cre
89643
Lenti-sgArid1a/Cre
89642
Lenti-sgApc/Cre
89641
Lenti-sgLkb1/Cre
66894
Oligos:
Universal Reverse Primer (5’ → 3’):
AGCTAGGGATCCGCCGCATAACCAGTG
Barcoded Forward Primer (5’ → 3’):
AGCTAGTCCGGXXXXXXXXAANNNNNTTNNNNNAANNNNNATGCCCAAGAAGAAGAGGAAGGTGTC
NOTE: 8-nucleotide ID specific to each individual sgRNA (sgID; X) and the 15-nucleotide random barcode (BC; N). sgID is optional, if you are not creating a pool of vectors, the second barcode may not be necessary for your experience.
Reagents:
- XmaI (NEB, R0180)
- BspEI (NEB, R0540)
- T4 DNA Ligase (NEB, M0202)
- PrimeSTAR® HS DNA Polymerase (premix) (Clontech, R040A)
- Qiagen® PCR Purification Kit (Qiagen, 28106)
- ElectroMAX DH10B cells (Thermo Fisher, 18290015)
- 0.1 cm GenePulser/MicroPulser Cuvettes (Bio-Rad, 165-2089)
- Qiagen® HiSpeed MidiPrep Kit (Qiagen, 12643)
- Qiagen® MinElute Gel Extraction Kit (Qiagen, 28604)
BD MicroPulser™ Electroporator (Bio-Rad,165-2100)
PCR amplify the barcoded insert off of Lenti-PGK/Cre plasmid.
Reaction mix:
200 l PrimeStarHS Mix
12 l Forward Primer (10 M)
12 l Reverse Primer (10 M)
8 l Template (500 ng/L)
168 l H20
400 l Total Split into 4, 100 l reactions
PCR Program:
Temp. (°C) Time (min)
- 98 1:00
- 98 0:15
- 55 0:05
- 72 1:00
- Go to step 2, 34x
- 72 5:00
4 forever
- Pool the PCR reactions and PCR purify using the Qiagen® PCR Purification Kit. Elute in 35 l H20.
- Digest barcoded insert with BspEI and BamHI. Reaction mix:
5 l Buffer 3.1
1.5 l BspEI
1.5 l BamHI
3 g Barcode Insert
H20 to 50 l
50 l Total Incubate at 37°C 2 hours
- Concurrently digest Lentiviral Plasmid with XmaI and BamHI. Reaction mix:
5 l Cutsmart Buffer
1.5 l XmaI
1.5 l BamHI
5 g Lentiviral Plasmid
H20 to 50 l
50 l Total Incubate at 37°C 2 hours
- Run out digested products on a 2% agarose gel and gel extract the fragments using the Qiagen® MinElute Gel Extraction Kit (Qiagen, 28604) and elute in 25 l of H20.
- Ligate digested barcoded insert into digested Lentiviral Plasmid Reaction mix:
8 l Ligation Buffer
1.6 l T4 DNA Ligase (NEB, M0202)
300 ng Digested Lentiviral Plasmid
180 ng Digested Barcoded Insert
H20 to 80 l
80 l Total Incubate at 16°C overnight
- PCR purify the ligation product using the Qiagen® PCR Purification Kit (Qiagen, 28106)
- Transform 1 l into 20 l ElectroMAX DH10B cells (Thermo Fisher, 18290015). Use 0.1 cm GenePulser/MicroPulser Cuvettes (Bio-Rad, 165-2089) and BD MicroPulser™ Electroporator (Bio-Rad,165-2100)at 1.9 kV.
- Ad 500 l of SOC media and shake at 200 rpm for 30 minutes at 37°C.
- Plate on LB-Amp plates (1x1 l, 1x 10 l, and 5x 100 l) and incubate at 37°C overnight.
- Count colonies on either the 1 l or 10 l plate and use these counts to estimate the colony number on the 100 l plates. The total number of colonies counted will provide an estimate of the number of unique barcodes obtained.
- Scrape bacterial colonies off of the LB-Amp plates in 10 mL of LB-Amp per plate.
- Pool liquid in a flask and shake at 200 rpm for 30 minutes at 37°C.
- Midi-prep the barcoded plasmid pool using the Qiagen® HiSpeed MidiPrep Kit (12643).
- Determine plasmid concentration and confirm barcoding by Sanger sequencing.
2-3 full days.
The transformation will be inhibited if there is any salt left in the transformed plasmid solution.
You may need to titrate the voltage on the electroporater, depending on the machine they behave differently.