Barcoding lentiviral Cre vectors for use in experiments involving downstream Tuba-seq analysis.

doi:10.1038/protex.2017.090

Method Article

Barcoding lentiviral Cre vectors for use in experiments involving downstream Tuba-seq analysis.

https://doi.org/10.1038/protex.2017.090

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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).

Molecular Biology

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.

None.

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Barcoding lentiviral Cre vectors for use in experiments involving downstream Tuba-seq analysis.

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