TECHNICAL MANUAL

SMARTvector™ LentiviralshRNA Pooled Libraries
SMARTvector™ Inducible LentiviralshRNA Pooled Libraries
shMIMIC Lentiviral microRNA
Pooled Libraries
shMIMIC Inducible LentiviralmicroRNA Pooled Libraries

Contents
1 Introduction....................................................................................................................................... 3
A. Available libraries................................................................................................................................................................................ 3
B. SMARTchoice™ configuration platform........................................................................................................................................ 4
C. Optimized pooled screening workflow..................................................................................................................................... 5

2 .Laboratory protocols and calculation tracking worksheet........................................................... 7
3 Required materials for lentiviral pooled screening....................................................................... 7
A. Materials required............................................................................................................................................................................... 7
B. Additional recommended materials............................................................................................................................................ 7

4		 Assay development and optimization............................................................................................ 8
A. Selection of the optimal promoter for your lentiviral pooled library............................................................................. 8
B. Optimization of lentiviral transduction....................................................................................................................................... 8
C. Determination of functional titer.................................................................................................................................................. 9
D. Optimization of puromycin selection....................................................................................................................................... 11
E. Determination of assay-specific screening conditions....................................................................................................... 12
F. Selection of average fold representation and number of biological replicates......................................................... 12
G. Calculation of number of cells needed for transduction................................................................................................... 12
H. Calculation of volume of lentiviral particles needed for transduction......................................................................... 145		 Primary screen................................................................................................................................. 15
A. Cell transduction and selection...................................................................................................................................................16
B. Genomic DNA isolation................................................................................................................................................................... 17
C. PCR amplification of constructs from genomic DNA.......................................................................................................... 17
i. Number of PCR reactions........................................................................................................................................................... 17
ii. Multiplexing of high-throughput sequencing samples................................................................................................20
iii. PCR from genomic DNA...........................................................................................................................................................21

6		 Illumina platform sequencing....................................................................................................... 227		 Hit identification and follow up..................................................................................................... 238		Appendix.......................................................................................................................................... 24
A. Laboratory protocols and calculation tracking worksheet...............................................................................................24
B. Key terms and definitions..............................................................................................................................................................28
C. Stability and storage........................................................................................................................................................................29
D. Quality assurance and control.....................................................................................................................................................29

9		 Frequently asked questions........................................................................................................... 30
10 References....................................................................................................................................... 33
11 Lentiviral particle product safety level information................................................................... 33
12 Label licenses................................................................................................................................... 35

SMARTvector Lentiviral shRNA &shMIMIC Lentiviral microRNA Pooled Libraries
Technical Manual

1

Introduction
SMARTvector Lentiviral shRNA and shMIMIC Lentiviral microRNA Pooled Libraries are pools of lentiviral constructs
expressing short hairpin RNAs (shRNAs) targeting coding genes for knockdown or mature microRNAs mimics for
microRNA-overexpression, respectively. Pooled screening can be performed to identify the function of coding or
microRNA genes in the regulation of cellular responses and signaling pathways without the need for costly automationand equipment required in arrayed screening.

A. Available libraries
Table 1 lists the pre-defined pooled lentiviral libraries that are available.
Table 1. Available SMARTvector Lentiviral shRNA and shMIMIC Lentiviral microRNA Pooled Libraries.

Human

Mouse

Number oftargeted genes4

Number of pools × numberof constructs per pool4,5

Number oftargeted genes4

Number of pools × numberof constructs per pool4,5Whole Genome

19 24116 pools × 9513 constructs

21745

18 pools × 9502 constructs

Druggable Genome

7341

6 pools × 9735 constructs9723

8 pools × 9670 constructs

GPCR

377

1 pool × 3012 constructs494

1 pool × 3909 constructs

Ion Channel

340

1 pool × 2709 constructs

332

1 pool × 2646 constructs

Protein Kinase702

1 pool × 5602 constructs

697

1 pool × 5558 constructs

Phosphatase245

1 pool × 1948 constructs

268

1 pool × 2141 constructs

Protease466

1 pool × 3702 constructs529

1 pool × 4206 constructs

Ubiquitin Conjugate

557

1 pool × 4431 constructs

511

1 pool × 4069 constructs

Number of mature microRNAs

Number of unique designs

Number of pools × numberof constructs per pool5

2580

2555

1 pool × 2665 constructs

1913

1896

1 pool × 2006 constructs

355 human + 341 mouse386

1 pool × 496 constructs

SMARTvector Lentiviral shRNA Pooled
Library1,2,3,6

shMIMIC Lentiviral microRNA
Pooled Library1,2,6
Human
Mouse
Human-Mouse Conserved

Libraries are available in constitutive promoters (hCMV, mCMV, hEF1α, mEF1α, CAG, PGK, UBC) with fluorescent reporters (TurboGFP, TurboRFP or
no reporter) or tetracycline-inducible promoters (TRE3G™) with fluorescent reporters (TurboGFP or TurboRFP). Some promoter options may only beavailable as custom products or upon request.

1

Constitutive and inducible lentiviral pooled libraries are provided at ≥ 5 × 108 TU/mL (± 20%) and ≥ 5 × 107 ± TU/mL (± 20%), respectively.

2

Additional gene families are available on the website and by request (ts.dharmacon@horizondiscovery.com).

3
4

The number of genes and constructs per pool are subject to change at any time without notice.

Number of constructs per pool includes gene-specific and negative control constructs.

5

Custom libraries targeting rat gene families or researcher-defined human, mouse or rat gene lists are available
by request (ts.dharmacon@horizondiscovery.com).

6

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SMARTvector Lentiviral shRNA Pooled Libraries include:
1.

shRNAs targeting coding genes in NCBI Reference Sequence Database: Up to eight shRNA designs per gene basedon our proprietary algorithm;

2.

Gene-specific controls: shRNA constructs targeting 34 protein-coding genes including ACTB, GAPDH, LAMB1, and PPIB
(eight constructs per gene, 272 shRNAs total) in each pool;

3.

Negative controls: 100 Non-targeting shRNA controls in each pool.

shMIMIC Lentiviral microRNA Pooled Libraries include:
1.

shMIMIC microRNAs (lentiviral microRNA expression constructs) representing all human or mouse mature microRNAs
as annotated in miRBase database;2.

Gene-specific controls: shRNA constructs targeting 10 protein-coding genes including ACTB, GAPDH, LAMB1, and PPIB
(eight constructs per gene, 80 shRNAs total) in each pool;

3.

Negative controls: 30 Non-targeting shRNA controls in each pool.

shMIMIC Lentiviral Human-Mouse Conserved microRNA Pooled Libraries include:
1.

shMIMIC Lentiviral microRNAs representing human and mouse mature microRNAs that are broadly conserved acrossmost vertebrates (www.targetscan.org), plus all matures that are identical between human and mouse.

2.

Gene-specific controls: shRNA constructs targeting five human and five mouse protein-coding genes including
human and mouse GAPDH and PPIB (eight constructs per gene, 80 shRNAs total) in each pool;

3.

Negative controls: 30 Non-targeting shRNA controls in each pool.

All libraries are delivered with a data file containing complete library information, including: gene IDs, construct
sequences, complete list of controls, and counts per millions of mapped reads.
Also available for use with screening libraries are optimized primers and experimentally tested protocols for reliable
identification of shRNA and microRNA hits by high-throughput sequencing on an Illumina platform (see Section 3 fordetails on additional recommended materials).

B. SMARTchoice™ configuration platform
The SMARTvector Lentiviral shRNA and shMIMIC Lentiviral constructs are part of the SMARTchoice configuration platform,
an innovative system ideally suited for RNA interference (RNAi). The purpose is to provide the researcher with the mosteffective tools for delivering and expressing genetic content in their cells of interest.
The SMARTchoice platform provides a choice of constitutive (Figure 1A) or tetracycline-inducible (TRE3G™; Clontech,
Mountain View, CA) promoters (Figure 1B), and presence or absence of reporter genes (TurboGFP or TurboRFP; Evrogen,Moscow, Russia).

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A.

hCMV
mCMV
hEF1α
mEF1α
PGK
CAG
UBC

SMARTchoicepromoters

5’ LTR

Ψ

RRE

or
or

tGFP IRES PuroRtRFP IRES

None

B.
TRE3G
5’ LTR

Ψ

RRE

or

tGFP

WPRE 3’ SIN LTR

SMARTvector universal scaffold

mCMV
hEF1α
mEF1α
PGK

SMARTchoicepromoters

PuroR 2A Tet-on 3G WPRE 3’ SIN LTR

tRFP
SMARTvector
universal scaffold

Vector element

UtilityhCMV

Human cytomegalovirus immediate early promoter

mCMV

Mouse cytomegalovirus immediate early promoter

hEF1α

Human elongation factor 1 alpha promoter

mEF1α

Mouse elongation factor 1 alpha promoter

PGK

Mouse phosphoglycerate kinase promoter

CAG

Human cytomegalovirus, chicken β-actin hybrid promoter

UBC

Human ubiquitin C promoter

TRE3G

Inducible promoter with Tetracycline Response Elements which is activated by the Tet-On 3G
protein in the presence of doxycyclinetGFP or tRFP

TurboGFP or TurboRFP reporter for visual tracking of transduction and expression

None

No reporter option for use in applications where fluorescence is not required

IRES

Internal Ribosomal Entry Site allows for translation initiation in the middle of a mRNA sequence

PuroR

Puromycin resistance permits antibiotic-selective pressure and propagation of stable integrants

SMARTvector universal scaffold

Universal primary microRNA context into which an shRNA or mature microRNA is embedded

5' LTR

5' Long Terminal Repeat necessary for lentiviral particle production and integration of the construct into the host cell genomeΨPsi packaging sequence allows lentiviral genome packaging using lentiviral packaging systems

RRE

Rev Response Element enhances titer by increasing packaging efficiency of full-length lentiviral genomes

WPRE

Woodchuck hepatitis Post-transcriptional Regulatory Element enhances transgene expression in target cells

3' SIN LTR

3' Self-inactivating Long Terminal Repeat for generation of replication-incompetent lentiviral particles

Figure 1. Maps of the SMARTvector constitutive (A) and inducible (B) lentiviral vectors with promoters available from the SMARTchoice configuration platform.

C. Optimized pooled screening workflow
Once vector configuration options and basic transduction and assay conditions have been optimized, screening begins
by transducing cells at a low multiplicity of infection (MOI) with a lentiviral pooled library (see Figure 2 for a screening
workflow diagram). Individual cells in the resulting transduced population will contain single constructs integrated
into their genomes. Following transduction, a selective pressure is applied such that those constructs involved in a
specific biological response can be identified. As a result of the selective pressure, cells expressing the shRNA or mature
microRNA construct are either enriched or depleted in the cell population.
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To identify "hits", genomic DNA (gDNA) is isolated from the initial transduced cell population (reference cells) and fromthe transduced cell population that remains following the application of selective pressure and/or phenotypic
selection (experimental cells). shRNA and microRNA constructs within the isolated gDNA are amplified using
SMARTvector Forward and Reverse Index PCR primers that have been designed and optimized to minimize amplification
bias. Following amplification, indexed reaction products can be directly loaded onto Illumina flow cells and sequenced
using SMARTvector Illumina Sequencing Primers. The differences in shRNA or microRNA construct abundance between
reference and experimental cell populations can then be determined to identify hits from the primary screen.

Assay development and optimization

Each step of the lentiviral pooled screening workflow, from transduction to hit identification, has been empirically tested.
Optimal PCR conditions were identified to ensure uniform amplification and high reproducibility. The screening system
for SMARTvector Lentiviral shRNA and shMIMIC Lentiviral microRNA Pooled Libraries utilizes universal Illumina-adapted
PCR primers for identification of shRNA or shMIMIC microRNA sequences from gDNA by high-throughput sequencing on
Illumina instrumentation. SMARTvector primers are designed
for efficient amplification of constructs while minimizing
Transduction parameters
Screening parameters
thermodynamic bias and variation in representation. In
addition, SMARTvector PCR primers havebuilt-in adaptor and index sequences that allow the researcher
to easily move from PCR amplification to Illumina
Selective
pressurehigh-throughput sequencing. Direct identification of
construct guide strands facilitates data analysis and ensuresaccurate target gene identification.

Transduce withlentiviral shRNA or microRNA pool
Figure 2. Pooled lentiviral shRNA or microRNA screening workflow.

Assay Development and Optimization: Establish optimal
experimental conditions, including those for lentiviral transduction
and screening parameters, such as selective pressure and time
between collection of reference and experimental samples.

Primary screen

Select transduced cells

Reference sample

Experimental sample
Selective pressure

Select phenotype

Log Experimental/Reference

Hit identification

Isolate gDNA and analyze
sequence of integrated shRNA
Enriched shRNAs
or microRNAs

Depleted shRNAs
or microRNAs
Log mean counts

Primary Screen: A stable population of cells expressing single
integrants of shRNA or microRNA constructs is created bytransducing a lentiviral pooled library at low MOI. Transduced
cells are split into reference and experimental populations forapplication of a selective pressure and/or phenotypic selection.
gDNA is then isolated from reference and experimental
populations of transduced cells. Illumina-adapted SMARTvector
PCR primers and Phusion™ Hot Start II High-Fidelity DNA
Polymerase are used to PCR amplify integrated constructs
including various control sequences and add Illumina flow-cellbinding sequences. The resulting amplicons are sequenced on
Illumina platform sequencers, using the sequencing primers
provided.
Hit Identification and Follow Up: Sequences in reference andexperimental samples are identified and compared. Constructs
that are enriched or depleted are identified as hits to be confirmed
and studied further using individual SMARTvector LentiviralshRNA or shMIMIC Lentiviral microRNA constructs in additional
phenotypic and/or biochemical assays.
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2

Laboratory protocols and calculation tracking worksheet
The Laboratory protocols and calculation tracking worksheet is a tool for the recording of key calculations andexperimental parameters (see Appendix A). Please review all of the detailed protocols provided in this manual prior tousing the worksheet. An electronic version of the Laboratory protocols and calculation tracking worksheet can also
be downloaded from the Dharmacon web page. The downloadable Excel worksheet allows users to incorporate specificinputs related to their screen and performs key calculations to simplify protocol planning.

3

Required materials for lentiviral pooled screening
A. Materials required•

Lentiviral pooled library (Table 1)•

Positive and/or negative controls for optimization with matched promoter/reporter options»»

Catalog numbers for gene-specific and negative controls with a constitutive promoter may be found here:
dharmacon.horizondiscovery.com/rnai/controls/shrna/smartvector-lentiviral-controls
Catalog numbers for gene-specific and negative controls with an inducible promoter may be found here:
dharmacon.horizondiscovery.com/rnai/controls/shrna/smartvector-inducible-lentiviral-controls•

SMARTvector Indexing PCR and Sequencing Primer Kit Set A (Illumina platform) (Dharmacon, Cat #PRM7668)•

SMARTvector Indexing PCR and Sequencing Primer Kit Set B (Illumina platform) (Dharmacon, Cat #PRM10186) for
additional indexed primers (if experimental design requires)•

Phusion Hot Start II High-Fidelity DNA Polymerase and 5x Phusion HF Buffer (Thermo Scientific, Cat #F549X;
NEB, Cat #M0535X).
Please see Section 4 to determine the amount of Phusion DNA polymerase required for a screening workflow.

B. Additional recommended materials•

For constitutive promoters, use the SMARTchoice Promoter Selection Plate (Dharmacon, Cat #SP-001000-01) for
optimal promoter selection•

For inducible vectors, use the SMARTchoice Inducible Non-targeting Control 4-Pack (Dharmacon, Cat #VSC6847)
for optimal promoter selection•

Doxycycline hyclate (required for inducible promoter options only; Thermo Scientific, Cat #ICN19895510)•

Polybrene (American Bioanalytical, Cat #AB01643)•

Betaine Solution 5 M (Sigma-Aldrich, Cat #B0300)•

PCR purification kit (QIAquick™ PCR Purification Kit, Qiagen, Cat #28104 or similar)•

Low range DNA Ladder (GeneRuler™ Low Range DNA Ladder, ready-to-use, 25-700 bp,
Thermo Scientific, Cat #SM1193 or similar)•

Genomic DNA purification kit (Blood and Cell Culture DNA Purification Kit, Qiagen, Cat #13362 or similar)

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4•

Nucleotide mix dNTP set, 10 mM each (GE Life Sciences, Cat #28-4065-64 or similar)•

HyClone™ Puromycin 2 HCl (GE Life Sciences, Cat #SV30075.01 or similar)•

Base Medium: antibiotic-free cell culture medium (without supplements or serum)•

Growth Medium: antibiotic-free cell culture medium (with serum and/or supplements) recommended formaintenance of the cells•

Transduction Medium: the base cell culture medium containing lentiviral particles (with transduction
additives and serum, if necessary)•

Selection Medium: Growth Medium supplemented with the appropriate concentration of puromycin•

Resazurin cell viability reagent or similar

Assay development and optimization
Please use the Laboratory protocols and calculation tracking worksheet (see Appendix A) as a guide.

A. Selection of the optimal promoter for your lentiviral pooled library
SMARTchoice Promoter Selection Plate for constitutive expression
The SMARTchoice Promoter Selection Plate (purchased separately) is designed to allow data-driven promoter selection
prior to ordering a SMARTvector Lentiviral shRNA Pooled Library or a shMIMIC Lentiviral microRNA Pooled Library.
This unique tool permits the simultaneous evaluation of seven different constitutive promoters (hCMV, mCMV, PGK,hEF1α, mEF1α, CAG, and UBC) in order to select one that is most effective in your cell line or cell type of interest.
Detailed protocols for adherent or suspension cells are described in the SMARTvector Lentiviral shRNA Technical Manual(dharmacon.horizondiscovery.com/uploadedFiles/Resources/smartvector-constitutive-manual.pdf).
SMARTchoice Inducible Non-targeting Control 4-Pack for inducible expression
SMARTvector and shMIMIC Inducible lentiviral vector configuration options include four RNA pol II promoter options:mouse CMV, PGK, human EF1α and mouse EF1α; all of which control the expression of puromycin resistance and the
Tet-On 3G transactivator. Use the SMARTchoice Inducible Non-targeting Control 4-Pack to evaluate which of thesepromoters are most active in the cells of interest. Consult the SMARTvector Inducible Lentiviral shRNA Technical Manual(dharmacon.horizondiscovery.com/uploadedFiles/Resources/smartvector-inducible-lentiviral-shRNA-manual.pdf) for
detailed protocols.

B. Optimization of lentiviral transduction
While lentiviral particles exhibit broad cell tropism, the conditions for successful and efficient delivery can varysignificantly. It is essential to determine the optimal lentiviral transduction conditions in each cell line or type of interest.
Please keep in mind that the conditions selected during these optimization steps must be compatible with primary
screening protocols and conditions. SMARTvector Non-targeting Control lentiviral particles can be used for optimizationof transduction conditions. Detailed protocols for transduction optimization are described in the SMARTvector LentiviralshRNA Technical Manual (dharmacon.horizondiscovery.com/uploadedFiles/Resources/smartvector-constitutive-manual.
pdf). For inducible lentiviral vector options consult the SMARTvector Inducible Lentiviral shRNA Technical Manual(dharmacon.horizondiscovery.com/uploadedFiles/Resources/smartvector-inducible-lentiviral-shRNA-manual.pdf).
Parameters that may influence the efficiency of lentiviral transduction include, but are not limited to:8

SMARTvector Lentiviral shRNA &shMIMIC Lentiviral microRNA Pooled Libraries
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Transduction medium: When possible, the transduction of cells with lentiviral particles should be performed in asmall volume of low-serum (0.5-2%) or serum-free medium. For cells sensitive to low serum conditions, transduction
optimization can be performed in complete medium.
Transduction duration: Incubation time can vary between 4 and 24 hours and will depend on the cells of interest.
Transduction medium additives: Cationic polymers such as hexadimethrine bromide (Polybrene) may be added toenhance lentiviral particle binding to the cell surface. We recommend testing a range of concentrations, from
0-10 μg/mL, for identification of optimal transduction efficiency with minimal or no cell toxicity. If Polybrene is toxic tothe cells of interest, DEAE-Dextran (1-10 μg/mL) or Protamine Sulfate (1-50 µg/mL) may be substituted.
Cell density at transduction: The density at which cells are seeded may also influence transduction efficiency. We
recommend seeding cells at a range of densities for optimization of transduction efficiency. Plate sizes for screening
should be chosen accordingly.

C. Determination of functional titer
Functional titer can be determined either by counting GFP or RFP-positive colonies using fluorescence microscopy
or by fluorescence activated cell sorting (FACS) analysis of GFP/RFP-positive cells. For no reporter lentiviral vector
options we suggest using a functional titration protocol such as limiting dilution with cell viability assay by crystal
violet staining (dharmacon.horizondiscovery.com/uploadedfiles/resources/titer-crystal-violet-protocol.pdf) or genomic
qPCR assay.1
Lentiviral transduction efficiency can vary by target cell type. The titer of SMARTvector Lentiviral shRNA particles
reported on the Certificate of Analysis (C of A) was measured in HEK293T cells, so the relative transduction efficiency of
your cell type will likely be different. Therefore, it is recommended that a relative functional titer is determined in yourcells using SMARTvector Non-targeting Control Particles and then used to convert the reported titer of each lentiviralpool for the primary screen.SMARTvector Non-targeting Control Particles are purchased separately and should bepromoter-matched to the pooled library.
Day 1:
1.

The day before transduction, seed a 96-well cell culture plate (Destination Plate) with your cells at the density
determined during transduction optimization. Grow cells overnight.

2.

Make dilution medium using serum, Polybrene conditions and cell medium determined during transduction
optimization. Make dilutions of SMARTvector Non-targeting Control Particles in a round-bottom 96-well plate(Dilution Plate). As shown in Figure 3 and Table 2, use one row of the plate for each replicate of the dilution series ofthe lentiviral stock. We recommend performing two replicates. The procedure for dilution of the lentiviral stock is
described below and results in a series of five-fold dilutions to reach a final of 390 625-fold dilution.
a. Add 40 μL of dilution medium to wells A1 and B1. Add 80 μL of dilution medium to each well A2-A8 and B2-B8.
b. Thaw SMARTvector Non-targeting Control Particles on ice and then add 10 μL each to wells A1 and B1. Mix
contents of each well by pipetting up and down 10-15 times. Discard pipette tip.
c. Transfer 20 μL from wells A1 and B1 to the corresponding wells in column 2. Mix contents of each well by
pipetting up and down 10-15 times. Discard pipette tip.
d. Repeat transfer of 20 μL for columns 3 through 8, mixing 10-15 times for each dilution.
e. Allow lentiviral particle:Polybrene complexes to form for 3-5 minutes at room temperature.
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3.

Remove culture medium from the cells in the 96-well plate Destination Plate.

4.

Transfer 25 μL of each dilution of lentiviral particles from the Dilution Plate to the corresponding wells in the
Destination Plate, being careful to not create bubbles.

5.

Incubate the cells for 4-24 hours (as determined during transduction optimization).

6.

Add 75 μL of normal growth medium to cells.

Days 4-5:
1.

Culture cells for 48-72 hours (as determined during transduction optimization).

2.

Choose one well in the Destination Plate for counting GFP/RFP-expressing colonies of cells. This should be awell in which individual colonies of cells can be visualized and counted. Count each multi-cell colony as one
transduction event, as the cells have been dividing over the culture period (Figure 4). Calculate the average
number of GFP/RFP-positive colonies from the same destination well of each replicate.

Make five-fold dilutions into
Dilution Plate according toprotocol and Table 2.
1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

A
B
C
D
E
F
G
H

Remove medium from
Destination Plate.
Add 25 µL diluted lentiviralparticles from each well in the
Dilution Plate to cells in thecorresponding well of the
Destination Plate
Dilution Plate

A
B
C
D
E
F
G
H
Destination Plate

Figure 3. Diagram fordilution series of lentiviralparticles (Dilution Plate)
and addition to cells(Destination Plate).

Table 2. Example setup for lentiviral particle dilution series.

Dilution Plate
Well

Lentiviral particle serial
dilution volume

Volume ofdilution medium

Dilution factor

A1
A2
A3
A4
A5
A6
A7
A8

10 µL (*control)
20 µL (from A1)
20 µL (from A2)
20 µL (from A3)
20 µL (from A4)
20 µL (from A5)
20 µL (from A6)
20 µL (from A7)

40 µL
80 µL
80 µL
80 µL
80 µL
80 µL
80 µL
80 µL

5
25
125
625
3125
15 62578 125390 625

Volume of diluted lentiviralparticles used in transduction(Destination Plate)
25 µL
25 µL
25 µL
25 µL
25 µL
25 µL
25 µL
25 µL

* SMARTvector Non-targeting Control Particles. Repeat identical dilution series in wells B1 to B8.

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Functional titer in transducing units per mL (TU/mL) can be determined using the following formula:
Number ofTurboGFPpositive
colonies×

Dilutionfactor(Table 2)÷

0.025(volume ofdiluted lentiviralparticles in mL)=

Functional titer of
SMARTvector Non-targeting
Control Particles stock inyour cell line (TU/mL)

Relative transduction efficiency of your cell type can be determined by using the following formula:

Figure 4. Example of individualcolonies in HEK293T cells 72 hours
post-transduction. Four colonies
are circled. Imaged at 40× magnification.

Functional titer of
SMARTvector Non-targeting
Control Particles
your cell line (TU/mL)

÷

Titer of SMARTvector
Non-targeting Control Particles stockreported on the C of A (TU/mL)=

Relativetransduction
efficiency of
your cell line

Use the calculated relative transduction efficiency of your cell line to calculate the relative
functional titer for each lentiviral pooled library using the following formula:
Relativetransduction
efficiency of
your cell line

×

Titer of the lentiviralpooled library
reported on the
C of A (TU/mL)=

Relativefunctional titer of thelentiviral pooled libraryin your cell line (TU/mL)

Calculation examples
If you counted 58 GFP/RFP-positive colonies in well A7 of the destination plate, the titer of the SMARTvector Non-targeting Control
Particles in your cell line would be calculated as follows:
58 (GFP/RFP positive colonies) × 78 125 (dilution factor) ÷ 0.025 (volume of diluted lentiviralparticles in mL) = 1.8 × 108 TU/mL functional titer of SMARTvector Non-targeting Control Particles in your cell line
If the titer for the SMARTvector Non-targeting Control Particles on the product insert was listed as 9.0 × 108 TU/mL, the relativetransduction efficiency of your cell type would be determined as follows:
1.8 × 108 TU/mL (functional titer in your cell line) ÷ 9.0 × 108 TU/mL (titer as indicated on productinsert) = 0.2 relative transduction efficiency
If the relative transduction efficiency of your cell line is 0.2 and the titer of a lentiviral pooled library, as indicated on the C of A,
is 5.0 × 108 TU/mL, the relative functional titer of the pool in your cell line would be determined as follows:
0.2 (relative transduction efficiency) × 5.0 × 108 TU/mL (titer as indicated on product insert) = 1.0 × 108 TU/mL relative
functional titer in your cell line

D. Optimization of puromycin selection
The SMARTvector lentiviral vector backbone contains the puromycin selectable marker which allows for selectionof cells that have integrated a SMARTvector shRNA or shMIMIC microRNA construct. Depending on your choice, the
SMARTvector lentiviral vector may also contain either the TurboGFP or TurboRFP fluorescent reporter protein to allow
selection by FACS to isolate cells expressing TurboGFP or TurboRFP. Prior to using puromycin selection to generate apurely transduced population of cells, it is important to determine the optimal concentration of puromycin required tokill non-transduced cells. This concentration can be identified by generating a puromycin kill curve.

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SMARTvector Lentiviral shRNA &shMIMIC Lentiviral microRNA Pooled Libraries
Technical ManualDay 1:
1.

Plate cells at a density appropriate for your cell type. Incubate overnight.

Day 2:
1.

Change to fresh medium supplemented with puromycin at a range of concentrations (0-10 µg/mL).
Incubate for 3-6 days.

Day 4-8:
1.

Approximately every 2-3 days replace with freshly prepared puromycin medium.

2.

Monitor the cells daily and visually observe the percentage of surviving cells. Optimum effectiveness should bereached in 3-6 days under puromycin selection.

3.

The optimal antibiotic concentration to use is the lowest concentration that kills 100% of the cells in 3-6 days fromthe start of antibiotic selection.

E. Determination of assay-specific screening conditions
The lentiviral pooled library screening workflow described here facilitates identification of genetic regulators of a rangeof biological processes. Variables to consider when planning screening conditions include, but are not limited to, assay
duration, conditions of selective pressure (for example, concentration or duration), method of phenotypic selection(for example, measuring viability, proliferation, surface marker expression or migration), and doxycycline application
parameters (if using inducible promoter options). We recommend that you optimize all assay conditions prior to
beginning a lentiviral pooled library screen using, if possible, either an shRNA or a shMIMIC microRNA construct known
to be involved in the phenotype(s) of interest.

F. Selection of average fold representation and number of biological replicates
A critical consideration of lentiviral pooled screening is the extent to which any given construct in a pooled librarywill be represented in the screen; in other words, the number of cells that contain an independent genomic
integration of any given construct or the number of biological replicates of each construct integration event andsubsequent phenotypic selection. High construct representation results in high reproducibility between biologicalreplicates and ensures that there is a sufficient window for detection of changes in construct representation after
phenotypic selection.2
Recommendations:•

Between 500 and 1000 independent integrations per construct•

At least two biological replicates• We have observed that construct fold representation has a greater impact on the ability to identify hits than the number of biologicalreplicates. Therefore, we recommend performing at least two biological replicates, while maintaining an average construct fold

• representation as high as is practical or technically feasible for your screen.

G. Calculation of number of cells needed for transduction
Once transduction optimization experiments have been performed and the assay-specific screening parameters
determined, cells need to be prepared for transduction. The calculations below outline how to determine the numberof cells required at the time of transduction.

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MOI is defined as the ratio of transducing units of lentiviral particles to target cells. At high MOIs, each cell will likely
be transduced by more than one lentiviral particle. Conversely, at low MOIs, the probability that any cell is transducedwith more than one lentiviral particle is low (Table 3). For lentiviral pooled screen, each cell will ideally express a single
construct. This ensures that the knockdown of a single gene (in the case of shRNA constructs) or over-expression ofa single microRNA (in the case of shMIMIC microRNA constructs) in a particular cell is responsible for the resulting
phenotype. Therefore, we recommend performing lentiviral transductions at an MOI ≤ 0.3.
Table 3. Poisson distribution showing the distribution of cells with
the indicated number of lentiviral integrations at various MOIs.

To calculate the number of lentiviral integration events required to achievea given construct representation, use the following formula:

Number of lentiviral integrants per cell
MOI

0

1

2

3

4

0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0

0.90
0.82
0.74
0.67
0.61
0.55
0.50
0.45
0.41
0.37

0.09
0.16
0.22
0.27
0.30
0.33
0.35
0.36
0.37
0.37

0.00
0.02
0.03
0.05
0.08
0.10
0.12
0.14
0.16
0.18

0.00
0.00
0.00
0.01
0.01
0.02
0.03
0.04
0.05
0.06

0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.01
0.01
0.02

Numberof constructs
in thelentiviral pool×

Desired foldrepresentation=

Desirednumber of cellswith lentiviral
integrations

To calculate the number of cells you will need per sample at the time oftransduction, first determine the proportion of cells with lentiviral integrants(Table 3), and then use the following formula:Desirednumber of cellswith lentiviral
integrations

At each MOI, the portion of cells having 0, 1, 2, 3, or 4 lentiviral
integrants is indicated. For example, at an MOI of 0.3, only 3% of
cells are predicted to contain more than one lentiviral integrant.

÷

Proportionof cells withlentiviral
integrations=

Number ofcells requiredat the time oftransduction

Calculation examples
If you have a pool of 1000 constructs and you wish to achieve a 500-fold average representation, the desired number of lentiviral
integration events would be calculated as follows:1000 (number of constructs) × 500 (average representation) = 5.0 × 105 lentiviral integration events
At an MOI of 0.3, approximately 25% of seeded cells will undergo at least one lentiviral integration event. Therefore, calculate thenumber of cells needed at the time of transduction as follows:
5.0 ×105 (lentiviral integration events required) ÷ 0.25 (proportion of cells that undergo at least one
integration) = 2.3 × 106 cells needed at the time of transduction

Plate sizes should be chosen such that the required number of cells can reach the optimum density for transduction,
as determined above, at the time of transduction. Prepare additional plates of cells for each pool and biological replicate
you wish to transduce. The calculations below outline how to determine cell plating requirements.
To determine the number of plates required for each biological replicate, use the following formula:
Number ofcells requiredat the time oftransduction

÷Cell density attransduction

Size ofplate (mm2)÷=

Number of plates
required per sample

To determine the total number of plates to seed per pooled RNAi library, use the following formula:

Number of plates
required per sample

×

Numberof biologicalreplicates=

Number ofplates required
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Table 4 provides examples of cell and plate numbers and plate sizes for various targeted construct fold representations,assuming a pool size of 1000 constructs and a cell line with optimal transduction density of 250 cells/mm2.
Table 4. Number of cells required at the time of transduction and cell plating recommendations for indicated average construct fold representation
assuming pools of 1000 constructs, MOI of 0.3, optimal transduction density of 250 cells/mm2 and an effective surface area on 100 mm plates of 7800 mm2.

Construct fold representation

Number of lentiviral
integrations required

Number of cells
required at transduction

Recommended cell plating500

5 × 105

2.3 ×106

1 plate × 100 mm

1000

1 ×10

4.5 × 10

2 plates × 100 mm

66

H. Calculation of volume of lentiviral particles needed for transduction
Before proceeding with the lentiviral pooled library screen, confirm that you have sufficient lentiviral particles to performthe screen at the desired fold coverage, given the relative transduction efficiency of your cell line and the number ofbiological replicates you wish to perform.
Follow the steps below to calculate the volume of lentiviral particles required for each pool. All pooled libraries aresupplied as multiple aliquots of 25 μL of lentiviral particles, at titers of ≥ 5 × 108 TU/mL for constitutive vectors and≥ 5 × 107 TU/mL for inducible vectors.
Table 5 provides examples of volume of lentiviral particles needed for various fold representations and relativetransduction efficiencies. Additional lentiviral particles can be ordered separately.

First, identify the number of lentiviral particles (or transducing units, TU) necessary for each biological replicate
based on the required number of cells at the time of transduction by applying the following formula:		
MOI

×

Number of cells required at
the time of transduction=

Required number oflentiviral particles (TU)

Next, determine the volume of lentiviral particles required based on the following formula:
Required number oflentiviral particles (TU)÷Functional titer in your cellline (TU/mL)=

Volume of lentiviral particles
per biological replicate (mL)

To determine the total volume of lentiviral particles required for each pool, use the following formula:
Volume of lentiviral particles
per biological replicate (mL)×

Number of
biological replicates=Volume of lentiviralparticles per pool (mL)

Calculation examples
For example, at an MOI of 0.3 and a 500-fold average construct representation at transduction, you will have prepared 2.0 × 106 cells at
the time of transduction. Based on the above formula, calculate the number of lentiviral particles needed:
0.3 (MOI) × 2.0 × 106 cells = 6.0 × 105 TU of lentiviral particles are required for transduction
If you have determined that you need 6.0 × 105 TU and you have a functional titer of 1.0 × 108 TU/mL in your cell line, you can calculate
the volume of lentiviral particles needed as follows:
6.0 × 105 TU ÷ 1.0 × 108 TU/mL = 0.006 mL of lentiviral particles
If you are performing two biological replicates, you can determine the total volume of lentiviral particles required per pool as follows:
0.006 mL of lentiviral particles × 2 biological replicates = 0.012 mL of lentiviral particles
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Table 5. Volume of lentiviral particles at 5 × 108 TU/mL in HEK293T cells required for the indicated fold representation and relative transduction efficiencies. The volume of lentiviralparticles is indicated for one pool of 1000 constructs with one biological replicate at an MOI of 0.3.

Construct fold representation

5

mary screen

Relative transduction efficiency
1

0.5

0.2

0.1

500

1.2 µL2.4 µL

6 µL

12 µL

1000

2.4 µL

4.8 µL

12 µL

24 µL

Primary screen
The following sections describe the primary screen workflow (Figure 5). Please use the Laboratory protocols
and calculation tracking worksheet (see Appendix A) as a guide.

Transduce withlentiviral pool

Select transduced cells

Reference sample

Experimental sample
Selective pressure

Select phenotype
Isolate gDNA

Reference gDNA

Experimental gDNA
Figure 5. Primary screen workflow.

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A. Cell transduction and selection
The experimental conditions described here, and in Figure 6, serve as a guide for performing lentiviral transductions.
However, the precise cell number and volume of lentiviral particles necessary to achieve the desired MOI and average
construct fold representation should be determined specifically for each cell line of interest and each intended screening
experiment, as outlined in Section 4.
Similarly, conditions should be clearly defined prior to starting the screen for application of selective pressure andselection of phenotype of interest.
12-18
hours

Seed cells

48-72
hours

Transduce
cells

3-6
days

Confirmtransduction*

Puromycin
selection

Screenspecific

Selective
pressure

Phenotypic
selection

Figure 6. Timeline of primary screening. *Timing of doxycycline induction varies by assay when using inducible promoter options.

Day 1:
1.

Seed cells in normal Growth Medium in the number of plates determined in Section 4.G The number of cells
seeded should be determined by extrapolating from the number of cells needed at the time of transduction and the
doubling time of your cell type. Incubate overnight.

Day 2:
1.

The next day, remove the medium and add optimized Transduction Medium (see Appendix) with the appropriate
amount of lentiviral particles (Section 4.H) so that the cells are just covered. If a single lentiviral pool will be added tomultiple plates, as determined in Section 4.G, divide the volume of lentiviral particles evenly among plates.

2.

After the appropriate transduction time (see Section 4.B), add additional Growth Medium to your cells such that thecells can be incubated for 48-72 hours.

Days 4-8:
1.

At 48-72 hours post-transduction examine the cells microscopically for the presence of fluorescent reporter
expression (if a reporter was chosen for the lentiviral vector); this will be the first indication as to the efficiencyof transduction. If using an inducible promoter option, induction by doxycycline for a minimum of 24 hours will benecessary to visualize the fluorescent reporter (See note below).
When using an MOI = 0.3 you should expect approximately 25% of the cells to express TurboGFP, or a 25% cell survival rate following
puromycin selection for no-reporter or inducible products prior to induction.

2.

Begin puromycin selection to remove non-transduced cells. Monitor the cells daily and observe the percentage offluorescent-positive cells (if a reporter was chosen for the lentiviral vector). Every 48-72 hours, replace with fresh
medium containing puromycin and passage cells as needed.

Use the appropriate concentration of puromycin and the minimum number of days required to kill non-transduced cells as determinedby a puromycin kill curve (see Section 4.D)

Once a pure population of transduced cells has been obtained (3-6 days), begin screening. Split cells into at least
two populations: one as a reference and another (experimental) for application of selective pressure and phenotypic
selection. To maintain your desired construct fold representation in the library at each cell passage, always retain at least
the number of cells that corresponds to the desired number of lentiviral integrants.
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B. Genomic DNA isolation
Following selection, gDNA should be isolated from control and experimental cell populations.
Isolation of gDNA from cells transduced with lentiviral pooled libraries has been optimized in the protocol below using Qiagen Blood
and Cell Culture DNA Maxi Kit; however, kits from other manufacturers may also be suitable.

1.

Collect cells by trypsinization for counting and gDNA isolation. To maintain your desired fold representation during
gDNA isolation, use at least the number of cells that corresponds to the desired number of lentiviral integrants. The
most accurate results can be obtained by counting cell number prior to gDNA isolation. Follow the manufacturer’s
protocol for purification of gDNA from cell cultures.
To ensure high DNA quality and yield, do not use more than the manufacturer’s recommended cell number and be
sure that your gDNA is fully solubilized. If multiple purification columns are required to maintain representation ofyour sample, combine gDNA isolations after elution.

It is important that you elute gDNA samples in EDTA-free buffer to prevent inhibition of subsequent PCR reactions.

2.

Quantify the isolated gDNA using a spectrophotometer and assess the DNA purity by measuring the ratio of theabsorbance at 260 and 280 nm (A260/280) and at A230 (A260/230). High-quality gDNA samples should have an A260/280 ratioof 1.8 to 2.0, indicating the absence of contaminating proteins, and an A260/230 ratio of > 2.0, indicating the absence ofother organic contaminants.

C. PCR amplification of constructs from genomic DNA
The PCR amplification and high-throughput sequencing workflow described here (Figure 7) has been designed to amplify
shRNA and shMIMIC microRNA constructs from gDNA without bias, such that differences in construct representation after
sequencing are due to enrichment or depletion that occurs during the primary screen. It is important to use adequate
template copies per construct in the PCR amplification step such that the average fold representation at transduction is
maintained. This ensures assay reproducibility and facilitates hit identification.2
The SMARTvector Indexing PCR primers provided have been designed for use with Illumina sequencing platforms. They are notcompatible with other high-throughput sequencing systems.

i. Number of PCR reactions
Calculate the amount of gDNA required to maintain construct fold representation, assuming a single lentiviral integration
event per cell (genome), using the following formula:

Number ofcells with lentiviral
integrants

×

6.6 × 10 -3
ng/genome(see note below)=

Mass of gDNA
required to maintainrepresentation ofeach construct

The calculation above assumes a diploid human genome. Many cell lines are not diploid and calculations must be adjusted accordingly.
For diploid mouse cell lines use 6.1 × 10 -3 ng/genome.

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Reference gDNA Experimental gDNA
Illumina-adapted
SMARTvector Forward
shRNA or microRNA
PCR Primer
Construct
Pro-viral
Sequence

Amplify constructwith Indexed Illumina
Adapted Primers

Illumina-adapted and
SMARTvector Reverse
Index PCR Primer

Indexed amplicon pool (237 bp)

Column purify
verify amplicon length
SMARTvector Read 1 Illumina
Sequencing Primer
Illumina
flow cell

SMARTvector Read Illumina
Sequencing Primer

Perform high-throughput sequencing
with SMARTvector Read 1 and Index
Read Illumina Sequencing Primers

Sequence Reads in FASTQ format
Read X: CTCAGATCAAAGTCTATAGCCTAAGTGAAGCCACAGATGTATAGACTTTG
Read Y: ACAGATCAGCTGTTGGAGTCAATAGTGAAGATGTAATCAATTTGAAGNNA
Read Z: ACAGGAGGTCAAGGAAAAGTGAAGCCAGCCACACAGATGTATTGATTTTC

Figure 7. PCR amplification and Illumina high-throughput sequencing workflow.

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We have optimized the PCR conditions to remain in the linear phase of log amplification using Phusion Hot Start II DNA
Polymerase. We recommend a maximum of 825 ng of gDNA per PCR; using more gDNA per PCR will inhibit the efficiencyof the reaction and will result in either failure of the PCR or biased amplification.
Calculate the number of PCR reactions required for each sample using the following formula:
Mass of gDNA
required to maintainrepresentation ofeach construct (ng)÷825 ng perreaction=

Number of PCR reactions
required to maintainrepresentation ofeach construct** Round up the value obtained to the next whole number)Table 6 gives an example of the input gDNA and number of PCR reactions required for various fold representations
with a pool of 1000 constructs.
Table 6. Input gDNA for PCR amplification and number of PCR reactions at indicated fold representation for a pool of 1000 constructs.

Construct fold representation

Input gDNA (μg)

Number of PCR reactions

500

3.3

4

1000

6.6

8

The calculations above are used to determine the number of PCR reactions required per sample. To determine the total numberof PCR reactions required for your screen use the following formula:
Number of PCR
reactions per sample

×

Number ofsamples per pool=

Number of PCR
reactions per pool

To determine the amount of Phusion DNA Polymerase required for each lentiviral pooled library use the following formula:
Number of PCR
reactions per sample

×4 units ofpolymerase per PCR=

Units ofpolymerase per poolCalculation examples
If you anticipate 5 × 105 cells with lentiviral integrants, the mass of gDNA required would be calculated as follows:
5 × 105 lentiviral integrants × 6.6 × 10 -3 ng/genome = 3.3 × 103 ng
The maximum amount of gDNA per PCR is 825 ng, therefore the number of PCR reactions necessary can be calculated as follows:
3.3 × 103 ng gDNA ÷ 825 ng/PCR = 4 PCR reactions
If you have four samples (reference and experimental samples in biological duplicates), then you need the following total numberof PCR reactions:
4 PCR reactions × 4 samples = 16 PCR reactions
To perform 16 PCR reactions you need the following amount of Phusion Hot Start II DNA Polymerase:
16 PCR reactions × 4 units of Phusion polymerase/PCR = 64 units of Phusion polymerase required per pool

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Most high-throughput sequencing platforms provide a sufficiently high number of sequence reads to allow multiplexingof samples in sequencing runs (running multiple samples in one lane). For more accurate hit identification, we
recommend obtaining a minimum of 1000 reads per construct. If the output of your sequencing platform is higher than
the number of reads required per sample, it may be possible to multiplex samples in the same run. To calculate themaximum number of samples that can be loaded per lane, it is advisable to assume a lower number of sequence reads
than the maximum obtained on an optimum sequencing run. We suggest estimating the number of reads by adjusting
the manufacturer's specifications by a factor of 0.7.
From this information the number of samples you can run per lane can be determined using thefollowing calculations:
Number of constructs
in lentiviral pool×1000 reads coverage
per construct=

Expected numberof reads per
sequencing lane×

0.7 (numberof reads
adjustment)

Output reads
required
per sample÷

Output reads
required per sample=

Number ofsample indices
per laneCalculation examples
If your pool contains 1000 constructs, then the number of output reads required per sample can be calculated as follows:1000 constructs × 1000 reads/construct = 1 million output reads required per sample
If you are using a sequencing platform with a projected output of 15 million reads per lane then the number of samples perlane can be calculated as:
1.5 × 107 reads per lane × 0.7 (number of reads adjustment) ÷ 1 × 106 output reads required
per sample = 10 samples per lane

Illumina-adapted SMARTvector Indexing PCR primers are provided as 50 μM forward and reverse indexed primers.
The index tags are shown in Table 7.
Table 7. The index tags on SMARTvector Indexing PCR primers are compatible with Illumina TruSeq LT indices.

Item

Index

Index sequence

Kit A
SMARTvector Reverse Index PCR Primer 2

2

CGATGT

SMARTvector Reverse Index PCR Primer 4

4

TGACCA

SMARTvector Reverse Index PCR Primer 5

5

ACAGTG

SMARTvector Reverse Index PCR Primer 6

6

GCCAAT

SMARTvector Reverse Index PCR Primer 7

7

CAGATC

SMARTvector Reverse Index PCR Primer 1212

CTTGTA

SMARTvector Reverse Index PCR Primer 13

13

AGTCAA

SMARTvector Reverse Index PCR Primer 14

14

AGTTCC

SMARTvector Reverse Index PCR Primer 15

15

ATGTCA

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Item

Index

Index sequence

SMARTvector Reverse Index PCR Primer 16

16

CCGTCC

SMARTvector Reverse Index PCR Primer 18

18

GTCCGC

SMARTvector Reverse Index PCR Primer 19

19

GTGAAA

SMARTvector Reverse Index PCR Primer 1

1

ATCACG

SMARTvector Reverse Index PCR Primer 3

3

TTAGGCKit B (Optional)

SMARTvector Reverse Index PCR Primer 88

ACTTGA

SMARTvector Reverse Index PCR Primer 9

9

GATCAG

SMARTvector Reverse Index PCR Primer 10

10

TAGCTT

SMARTvector Reverse Index PCR Primer 11

11

GGCTAC

SMARTvector Reverse Index PCR Primer 20

20

GTGGCC

SMARTvector Reverse Index PCR Primer 21

21

GTTTCG

SMARTvector Reverse Index PCR Primer 22

22

CGTACG

SMARTvector Reverse Index PCR Primer 23

23

GAGTGG

SMARTvector Reverse Index PCR Primer 25

25

ACTGAT

SMARTvector Reverse Index PCR Primer 27

27

ATTCCTiii. PCR from genomic DNA
Once you have determined the number of PCR reactions you need per sample and your multiplexing capabilities, PCR
can be performed. Sequence amplification from gDNA has been optimized using Phusion Hot Start II DNA Polymerase.
The amplification conditions recommended have been determined to provide maximum yields of PCR product whilestaying within the linear phase of log amplification.
1.

Perform PCR in 50 μL reactions using 96-well PCR plates. PCR conditions have been optimized to amplify 825 ng
of gDNA per 50 μL reaction. As described above, calculate the number of reactions needed for the amplification of
total gDNA necessary to maintain the fold representation used at transduction. Table 8 provides the components
and volumes needed for one 50 μL PCR amplification reaction.

Table 8. PCR components for amplification of genomic DNA.

Component

Volume per reaction

Final concentration

5x Phusion HF Buffer

10 μL

1x

10 mM dNTPs

1 μL

200 μM each

SMARTvector Forward PCR Primer (50 μM)

0.5 μL0.5 μM

SMARTvector Reverse Index PCR Primer (50 μM)

0.5 μL0.5 μM

5 M betaine

5 μL

0.5 M

gDNA + PCR grade H2O

31 μL~ 825 ng/reaction

Phusion Hot Start II DNA Polymerase (2 U/μL)

2 μL

0.08 U/μL

Total

50 μL

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2.

To PCR amplify the SMARTvector or shMIMIC constructs, use the cycling conditions outlined in Table 9. The
conditions indicated in this protocol ensure that amplification is in the exponential phase when using Phusion Hot
Start II DNA Polymerase; if using another polymerase, the optimal cycle conditions should be determined empirically.
Table 9. PCR cycle parameters

3.

Time

98 °C

3 minutes

98 °C

10 seconds

60 °C

15 seconds

72 °C

15 seconds

72 °C

5 minutes

23 cycles

Temperature

Confirm that a 237 base pair amplicon is obtained from each sample by running 10 μL of PCR product on
a 2% agarose gel.
Due to the low number of PCR cycles, the product will be a faint band on a gel before purification.

4.

Combine reactions amplifying the same gDNA sample into a single 1.5 mL tube. Purify the combined PCR amplicons
for each gDNA sample using a PCR purification kit.

Please be aware that gDNA often co-purifies with the PCR products. Although contaminating gDNA does not interfere with thenext-generation sequencing, it does affect the quantification of the amount of PCR amplicons by spectrophotometric or fluorimetricmethods. Therefore, it is recommended to evaluate the concentration of each amplicon using quantitative PCR or chromatographic
methods such as analysis on an Agilent Bioanalyzer System. Alternatively, the amplicons can be purified by an agarose gelelectrophoresis protocol and quantified by a fluorimetric DNA quantification method, such as QuBit™ (Life Technologies).

6

Illumina platform sequencing
Please use the Laboratory protocols and calculation tracking worksheet (see Appendix A) as a guide.
Manufacturer’s instructions should be followed for Illumina platform sequencing including (a) quantifying the libraryusing recommended quality standards and (b) dilution and denaturation of the library.
For the Illumina MiSeq Desktop Sequencer, we recommend spiking the library with 10% PhiX Control to serve as aninternal control and loading the library onto the flow cell at 7 to 10 pM using standard loading volumes. After loading
your sample into the Illumina MiSeq Reagent Cartridge, add 5 μL of the 100 μM SMARTvector Read 1 Sequencing Primer
stock to the "Read 1 Primer mix" reservoir and 5 µL of the 100 µM SMARTvector Index Read Sequencing Primer stock to
the "Index Primer Mix" reservoir on the Illumina MiSeq reagent cartridge (please check the MiSeq System User Guide
to confirm the inlet positions).
PhiX Control contains a balanced representation of A, T, G and C nucleotides helping with sequencing unbalanced or low diversity
libraries that may result during a screen.

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For the Illumina HiSeq 2500 system, we recommend spiking the library with 10% PhiX Control to serve as an internal
control and loading the library onto the flow cell at 6 pM for Rapid Run with onboard clustering or 7.5 pM for High Output
Run with c-bot clustering using standard loading volumes. Dilute the SMARTvector Read 1 Sequencing and SMARTvector
Index Read Sequencing primers into the respective Illumina primer reagents to a final concentrationof 0.5 µM.
At least 22 base single-end reads are required to sequence the guide strand of the SMARTvector shRNA or shMIMIC
microRNA, as outlined in Figure 8.
The SMARTvector Reverse Index PCR Primers for Illumina platforms use the same index sequences as the Illumina TruSeq LT Index
sequences, A001 to A027 (see Table 7).

SMARTvector Read1
Illumina Sequencing
Primer

50 baseread

22 basesguide ormaturestrand7

SMARTvector Index Read
Illumina Sequencing
Primer

13 bases 15 bases
loop

Passenger
strandFigure 8. Position of SMARTvector Sequencing
primers and contents of 50 base sequencing
reading including the shRNA guide, loop and partof the passenger strand or the shMIMIC microRNA
expression construct mature strand, loop and partof passenger strand.

Hit identification and follow up
Please use the Laboratory protocols and calculation tracking worksheet (see Appendix A) as a guide.
Alignment of sequence files and hit identification can be performed efficiently using open source programs that
require some bioinformatics knowledge. We recommend that you consult with an expert in bioinformatics for data
analysis and hit identification. A more detailed protocol can be downloaded from the SMARTvector Lentiviral shRNA
Pooled Library or the shMIMIC Lentiviral microRNA Pooled Library under Resources (dharmacon.horizondiscovery.com/
uploadedFiles/Resources/lentiviral-pooled-library-bioinfomatic-analysis-protocol.pdf). The first 21 to 22 bases of eachread correspond to the sequence of the guide strand of shRNA constructs or the mature strand of shMIMIC microRNA
constructs and are sufficient for differentiating among constructs. The remaining bases in the read include the loop
and a partial passenger strand sequence and can be ignored in the subsequent analysis (Figure 8). Using an alignment
tool, such as Bowtie 2,3 align the first 22 bases of each read to the reference file provided with your lentiviral pooled
library. Bowtie 2 has an option to ignore bases on the 3' end of the read; use this option to align only the first 22 basesof the guide strand. Using the output of the aligner, count the number of alignments for each construct. Next, use a
differential expression tool built for discrete count data, such as DESeq,4 to determine primary hits. Hits can be confirmed
and studied further using individual constructs that can be ordered from the SMARTvector Lentiviral shRNA or shMIMIC
Lentiviral microRNA collections.
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8

Appendix
A. Laboratory protocols and calculation tracking worksheet
An electronic version of this worksheet is available for download from the Dharmacon web page under the Resources
section (Pooled Screening Protocol Tracking Worksheet).

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B. Key terms and definitions
Antibiotic Drug Selection SMARTvector Lentiviral shRNA and shMIMIC Lentiviral miRNA constructs contain thepuromycin resistance gene that allows selection of transduced cells. Following transduction, cells can be selected andclonal populations can be derived.
Puromycin Resistance Gene (PuroR) The puromycin resistance gene is a mammalian selectable marker gene thatblocks the ability of the antibiotic puromycin to inhibit protein synthesis through ribosomal binding.
Multiplicity of Infection (MOI) Multiplicity of infection is the ratio of lentiviral transducing particles to cells. An MOI of
10 indicates that there are 10 transducing units (TU) for every cell in the well. It is important to note that different cell
types require different MOIs for successful transduction.
Polybrene Hexadimethrine bromide, also referred to as Polybrene, has been shown to enhance transduction of
mammalian cells two- to ten-fold by binding to the cell surface and neutralizing surface charge.
Transduction Efficiency This term refers to the fraction of cells that successfully integrate one or more lentiviralgenomes during the transduction process.
Transducing Unit Unit of measure that refers to the number of lentiviral particles that can transduce a population ofcells and integrate into the host genome. Titers are expressed as transducing units per milliliter (TU/mL).
Lentiviral Titer This is the number of transducing units (TU) present in a given volume (1 mL). Titer can be assessed by anumber of techniques including antibody- and PCR-based methodologies, flow cytometry and observation of transgene
expression following transduction of cells with serial dilutions of lentiviral particles.
Long Terminal Repeat (LTR) The long terminal repeat is the control center for retroviral gene expression. All of therequisite signals for gene expression are found in the LTRs: enhancer, promoter, transcription initiation (capping),
transcription terminator and polyadenylation signal. Expression directed by the viral LTR signals is carried out entirely by
host cell enzymes (RNA polymerase II, poly A synthetase, guanylyl transferase).
Self-Inactivating (SIN) Vector Self-inactivating lentiviral vectors are constructed by deleting the transcriptional
enhancers and/or the enhancers and promoter in the U3 region of the 3' LTR. After one round of vector replication,these changes are copied into both the 5' and the 3' LTRs, producing inactive lentiviral particles that are replicationincompetent.
Vesicular Stomatitis Virus Glycoprotein (VSVg) VSVg is an envelope protein derived from vesicular stomatitis lentiviralparticles (VSV), which has previously been shown to provide broad tropism.
Precursor microRNA (pre-miRNA) A stem-loop RNA resulting from microprocessor complex processing of the primary
microRNA transcript; precursors are shuttled out of the nucleus into the cytoplasm for further enzymatic processing
by Dicer to yield mature microRNAs.
Primary microRNA (pri-miRNA) The capped and polyadenylated RNA product of transcription of a microRNA gene

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Technical Manualin the nucleus, which contains local hairpin structures with imperfect base-pairing through the stem and, throughenzymatic processing by the microprocessor complex, yields the precursor microRNA.

C. Stability and storage
Lentiviral pooled libraries are shipped on dry ice as 25 μL aliquots and must be stored at -80 °C. Under these conditions,lentiviral particles are stable for at least 12 months. Repeated freeze-thaw cycles should be avoided,
as this is expected to negatively affect titer. When setting up a screening experiment using lentiviral pooled library
reagents, lentiviral particles should be thawed on ice, divided into smaller aliquots (if necessary) and immediately
returned to -80 °C.

D. Quality assurance and control
SMARTvector Lentiviral shRNA and shMIMIC Lentiviral microRNA Pooled Libraries are subject to stringent quality control
at multiple steps during the manufacturing process, including:1.

Evaluation of construct representation by high-throughput sequencing on Illumina sequencing platform:
Lentiviral pool master library glycerol stocks are sequenced using 2 × 100 base reads, filtered by PHRED score > 30,
and > 1000-fold read coverage. Pools have greater than 95% of the constituent constructs recovered. Greater than
80% of the guide strand sequences are perfect. The library has high standards for uniform construct distribution with
70% and 90% of the population within 10-fold and 25-fold of each other, respectively. A spreadsheet data file withconstruct annotation, sequence information and counts per million of mapped reads is provided with each screening
library.

2.

Lentiviral titering using quantitative PCR (qPCR) after transduction in HEK293T cells for functional titers.

3.

Confirmation of fluorescent reporter expression (TurboGFP or TurboRFP) when present by fluorescence microscopy
following transduction in HEK293T cells.

4.

Examination of each batch to ensure preparations are free from mold and bacterial contamination.

5.

A Certificate of Analysis (C of A) with specified lentiviral titers is included with each shipment.

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SMARTvector Lentiviral shRNA &shMIMIC Lentiviral microRNA Pooled Libraries
Technical Manual9Frequently asked questions
How many screenings can I perform with my pools?
The number of screenings which can be performed per lentiviral pool will depend on:1) The transducibility of your specific cell line/cell type;2) The fold representation you choose to maintain throughout the screening; and
3) The number of biological replicates you intend to include.
Is target gene down-regulation by the SMARTvector Lentiviral shRNA or shMIMIC Lentiviral microRNA
transient or permanent?
Lentiviral constructs, by way of lentiviral particle delivery of the engineered vector, integrate their payload into thehost cell genome. When cells are maintained under puromycin selection and as they divide, each recombinant genome
is replicated and transferred to the daughter cell progeny and can permanently express the RNAi construct (shRNAor shMIMIC microRNA). Thus, expression of the RNAi construct is stable and permanent in these cells. However, RNAibiology is complex and long-term down-regulation of putative gene targets depends on whether or not there is
redundancy or involvement in feedback loops. Also, cell-dependent epigenetic regulatory mechanisms may affect thelong term expression.
How do I know if my target cell type can be transduced with a SMARTvector Lentiviral shRNA or shMIMIC
Lentiviral microRNA construct?
SMARTvector and shMIMIC lentiviral constructs are pseudotyped with the VSVg envelope protein to allow broad tropism
across many cells. Therefore, most cell types are transduced, albeit, with varying degrees of efficiency. However, theefficiency of target gene regulation is not only a factor of successful transduction, but is also dependent upon theefficiency of transgene expression; this can be assessed by utilizing the SMARTchoice Promoter Selection Plate (Cat #SP001000-01) with the cells of interest. This will allow the simultaneous evaluation of seven SMARTchoice promoters.
Can SMARTvector Lentiviral shRNA or shMIMIC Lentiviral microRNA particles be further propagated in the lab?
No. SMARTvector and shMIMIC lentiviral constructs are engineered for biosafety and are provided as replicationincompetent lentiviral particles.
Where can I find the sequences of the mature microRNAs?
We provide mature microRNA sequences in the library data file, provided on a USB drive which was shipped with yourpooled library. You can also download mature microRNA sequences used to design the shMIMIC Lentiviral microRNAs
from the miRBase database website: ftp://mirbase.org/pub/mirbase
Can I use the SMARTchoice Promoter Selection Plate to optimize all of the transduction conditions for mycells of interest?
No. The SMARTchoice Promoter Selection Plate is not meant to be used to establish fundamental lentiviral transduction
conditions, such as optimal cell density, in your cells of interest. The purpose of the Promoter Selection Plate is to visuallyassess relative promoter activity. Basic experimental conditions (such as cell density, whether or not to include serum,
Polybrene concentration) should first be established prior to using the Promoter Selection Plate. The Promoter Selection
Plate contains duplicate wells of serially diluted lentiviral particles representing each promoter so that two separate30

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conditions can be tested simultaneously. After identification of the most active promoter for your cells of interest,
additional transduction optimization should be performed using control lentiviral particles.
I think I know the best promoter for my cells of interest. Do I still need to evaluate promoter activity with the
SMARTchoice Promoter Selection Plate or SMARTchoice Inducible Non-targeting Control 4-pack?
Promoter activity varies in different cell lines and cell types. This may affect the expression of the shRNA or shMIMIC
microRNA construct and, subsequently, the level of target gene regulation that can be achieved. You may choose thepromoter option that has been demonstrated, either by your own experimental observations or through referencesin the published literature, to actively express a transgene in your cells of choice. However, for optimal experimental
confidence we recommend selecting the best promoter empirically using the SMARTchoice Promoter Selection Plate
or Inducible Non-targeting Control 4-pack.
I have used the human CMV promoter previously for my mouse cell line with low performance. Will a different
promoter increase shRNA or shMIMIC microRNA function?
Poor performance may be due to low transduction efficiency or suboptimal promoter activity. Optimization oftransduction conditions and using promoters with optimal activity in your cells are essential to successful gene
regulation. Promoter activity varies in different cell lines and cell types, and we highly recommend using the
SMARTchoice Promoter Selection Plate or SMARTchoice Inducible Non-targeting Control 4-pack to determine themost active promoter in your cells of interest. For example, the mouse CMV promoter is very active in NIH/3T3 cells
and results in greater gene silencing compared to the same construct expressed from the human CMV promoter inthis cell line.
I am using human cell lines in my research; should I always choose a human promoter for best activity?
In some instances promoter activity correlates with the species from which it is derived. However, promoter activity does
not always follow a species-specific expression pattern. For example, we have observed mouse promoters to be the mostactive in some human cell lines, whereas both human and mouse promoters were most active in some rat cells. Choosing
the most effective promoter in a particular cell line is not always predictable, and therefore should be determined
empirically.
What quality control is performed on SMARTvector Lentiviral shRNA and shMIMIC Lentiviral microRNA
Pooled Libraries?
SMARTvector and shMIMIC lentiviral pools undergo multiple levels of analysis throughout production to ensure thequality of the final product. Pooled library glycerol stocks are subjected to high-throughput sequencing using 2 × 100
base reads, filtered by PHRED score > 30, and > 1000-fold read coverage. Pools have greater than 95% of the constructs
recovered. Greater than 80% of the guide strand sequences are perfect. The library has high standards for uniform
construct distribution with 70% and 90% of the population within 10-fold and 25-fold of each other, respectively. A
data file with NGS QC metrics is provided with each screening library. All components of the manufacturing process arecarefully monitored for bacterial and/or fungal contamination. Following packaging, lentiviral particles are titered byqPCR.
Are Dharmacon lentiviral particle products safe to use in the laboratory? What precautions should be taken
when handling lentiviral particles?
Lentiviral delivery systems have been employed in many research laboratories around the world without incident.

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SMARTvector Lentiviral shRNA &shMIMIC Lentiviral microRNA Pooled Libraries
Technical ManualHandling of lentiviral products requires extensive experience with cell culture techniques. It is vital that the protocols
provided by Dharmacon and the safety guidelines described for appropriate handling and storage are fully understoodand followed precisely (see Section 11, Lentiviral particle product safety level information).
How are Dharmacon lentiviral particle products manufactured?
High-titer lentiviral particle products are manufactured using the Trans-Lentiviral Packaging System to achieve high titers
as well as maximal biosafety. The transfer vector plasmid encoding the small RNA and the Trans-Lentiviral Packaging
plasmids are co-transfected into the HEK293T cell line. Subsequently, lentiviral particles are harvested, concentrated andpurified. All Dharmacon lentiviral particles are manufactured under stringent quality control
guidelines (see Appendix).
How are lentiviral particle products shipped?
Lentiviral particle products are shipped on dry ice for overnight domestic delivery or priority international fordelivery outside of the U.S.
How should lentiviral particle products be stored?
All lentiviral particle products must be stored at -80 °C. If necessary, the particles can be aliquoted upon the first thaw
to convenient volumes and the aliquots stored at -80 °C to minimize the number future freeze-thaws. However, werecommend avoiding multiple freeze-thaw cycles as much as possible.
What is the shelf-life of Dharmacon lentiviral particle products?
Lentiviral particles can be stored at -80 °C for at least 12 months without loss of titer. Our internal stability testing has
shown that if stored properly undisturbed, lentiviral particles can be used years after being produced without significant
loss of titer. Each thaw can reduce titer by an order of magnitude, so multiple freeze-thaws should be avoided as much as
possible.
Can lentiviral particle products be kept at 4 °C?
No. Once thawed, lentiviral particle preparations begin to decrease in titer. We advise against storing any lentiviral particles
at 4 °C. All lentiviral particle products must be kept frozen and stored at -80 °C until transductions are performed.
Where can I find titer information for my pools?
The titer of your lentiviral pools will be indicated on the Certificate of Analysis (C of A). C of As for purchased SMARTvector
Lentiviral shRNA and shMIMIC Lentiviral microRNA Pooled Libraries can also be requested from Technical Support.
Where can I find the sequence of an individual constructs included in my pool(s)?
The insert sequences are in the material sent with your shipment. Please contact Technical Support with your Purchase
Order number if you did not receive this material in your shipment.
How is gDNA input calculated to maintain desired construct fold representation?
The quantity of gDNA required for maintaining a desired fold representation can be calculated using the formulas andvalues (Table 10) below.

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SMARTvector Lentiviral shRNA &shMIMIC Lentiviral microRNA Pooled Libraries
Technical ManualThis calculation is for a diploid genome. Many cell lines are not diploid and calculations should be adjusted accordingly.

Table 10. Constants needed for calculating gDNA input.

Constants

Value

Mass of a base pair

660 g/mol/bp

Base pairs per diploid human genome

6 × 109 bp

Base pairs per diploid mouse genome

5.6 × 109 bp

Avogadro constant

6.02 × 1023 mol-1

Example:Calculate the mass of human genome:(6 × 109 bp/genome) × (660 g/mol/bp) = 4 × 1012 g/mol/genome(4 × 1012 g/mol/genome) ÷ (6.02 × 1023 mol-1) = 6.6 × 10 -12 g/genome

10 References
1.

R.H. Kutner, X.-Y. Zhang, and J. Reiser. Production, concentration and titration of pseudotyped HIV-1-based lentiviral
vectors. Nat. Protoc. 4, 495-505 (2009).

2.

Ž. Strezoska, et al. Optimized PCR conditions and increased shRNA fold representation improve reproducibility ofpooled shRNA screens. PLoS One. 7, e42341 (2012).

3.

B. Langmead and S. Salzberg. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 9, 357-359 (2012).

4.

S. Anders and W. Huber. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

11 Lentiviral particle product safety level information
This Lentiviral Particle Product Safety Level Information constitutes Product Documentation according to clause 1of the Product Terms and Conditions. It is applicable to the following lentiviral particle products:
SMARTvector™ Lentiviral shRNA
SMARTvector™ Inducible Lentiviral shRNA
shMIMIC Lentiviral microRNA
shMIMIC Inducible Lentiviral microRNA
SMARTchoice™ Promoter Selection Plate
SMARTvector™ Non-targeting Control
SMARTvector™ Inducible Non-targeting Control
SMARTvector™ Lentiviral shRNA Pooled Library
SMARTvector™ Inducible Lentiviral shRNA Pooled LibraryshMIMIC Lentiviral microRNA Pooled LibraryshMIMIC Inducible Lentiviral microRNA Pooled Library33

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Any investigator who purchases Dharmacon lentiviral particle products is responsible for consulting with their institution’s healthand biosafety personnel for specific guidelines on the handling of lentiviral vector particles. Furthermore, each investigator is fully
responsible for obtaining the required permissions for research use and the acceptance of replication-incompetent SIN lentiviral
vectors and replication-defective lentiviral particles into their local jurisdiction and institution.

The Products are solely for internal research use (as set forth in the Product Terms and Conditions) in laboratories where
the containment measures stated below and in applicable laws and regulations are met. Products may not be used for
diagnostic, therapeutic or other commercial purposes and may not to be administered to humans for any purpose or toanimals for therapeutic purposes. The Products are replication-incompetent, self-inactivating (SIN) and non-pathogenic(do not cause infectious human disease).
For questions concerning the design or production of the products, please contact our technical support team.
Dharmacon™, A Horizon Discovery Group Company
2650 Crescent Drive
Lafayette, CO 80026 USA
Tel: +1 800 235 9880; +1 303 604 9499
Fax: +1 800 292 6088; +1 303 604 9680
Email: ts.dharmacon@horizondiscovery.com (North America) or ts.dharmacon@horizondiscovery.com (Europe)
In the US:
For US guidance on containment for lentiviral vectors, please refer to:
1.

The Recombinant DNA Advisory Committee (RAC) guidelines for research with lentiviral vectors(osp.od.nih.gov/office-biotechnology-activities/biosafety/biosafety-guidance).

2.

The U.S. Department of Health and Human Services Centers for Disease Control and Prevention and National
Institutes of Health, Biosafety in Microbiological and Biomedical Laboratories (BMBL);

3.

The NIH Guidelines For Research Involving Recombinant DNA Molecules (NIH Guidelines),(osp.od.nih.gov/office-biotechnology-activities/biosafety/nih-guidelines).

In the EU:
For the EU directives, please consult the following:
1.

Council Directive 2009/41/EC of the European Parliament and of the Council of 6 May 2009 on the contained use ofgenetically modified micro-organisms. (revised version of Directive 90/219/EEC of the European Parliament and of
the Council of 23 April 1990 on the contained use of genetically modified micro-organisms, amended by Council
Directive 98/81/ EC of 26 October 1998); and2.

Council Directive 2001/18/EC of the European Parliament and of the Council of 12 March on the deliberate release
into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC.

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In Germany:
Required Containment Measures: The containment requirements as stated in the German Genetic Safety Ordinance(Gentechnik-Sicherheitsverordnung) of Safety Level 2* or higher have been assigned to the handling of the abovementioned lentiviral vector particles. Please note a higher Security Level might be required if the lentiviral vector
particles are used for genetic engineering operations with other products which require a higher Security Level. *Safety
Level 2: activities of low risk for human health and the environment by the state of scientific knowledge(Stand der Wissenschaft).
For the German regulations, please consult the following:
1.
2.

German Genetic Engineering Act (Gentechnikgesetz - GenTG); andGenetic Engineering Safety Ordinance (Gentechnik-Sicherheitsverordnung - GenTSV).

12 Label licenses
The shRNA and gene expression Products, use and applications, are covered by pending and issued patents. Certain
Label licenses govern the use of the products, these can be found at Dharmacon Licensing Statements. It is each Buyer’s
responsibility to determine which intellectual property rights held by third parties may restrict the use of Products for aparticular application. Please review the Label Licenses governing all use of the shRNA and gene expression Products.

If you have any questions contact

Illumina, HiSeq, MiSeq, and TruSeq are trademarks or registered trademarks of Illumina, Inc. Limited permission to copy or distributesequence information. Oligonucleotide sequences 2007-2011 Illumina, Inc. All rights reserved. Derivative works created by Illumina customers
are authorized for use with Illumina Instruments and products only. All other uses are strictly prohibited. Phusion is a trademark of Thermo
Fisher Scientific, Inc. TurboGFP and TurboRFP are trademarks of Evrogen Inc. ©2018 Horizon Discovery Group Company—All rights reserved.
All trademarks are the property of Horizon Discovery Company unless otherwise specified. UK Registered Head Office: Building 8100, Cambridge
Research Park, Cambridge, CB25 9TL, United Kingdom.

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V7-1218

t +44 (0) 1223 976 000 (UK) or +1 800 235 9880 (USA); +1 303 604 9499 (USA)f +44 (0)1223 655 581
w horizondiscovery.com/contact-us or dharmacon.horizondiscovery.com/service-and-support
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom