application Note

Application Workstations Automated High Throughput : Authors Stephen Hurt, PhD Streamlining Lentiviral RNAi PerkinElmer, Inc. Waltham, MA 02451 Screening Workflows on the USA JANUS Automated Workstation Hanh Le Broad Institute Cambridge, MA 02142 USA

Introduction Cultured and primary cell-based assays are now ubiquitous laboratory techniques used in drug discovery and basic research laboratories worldwide. With the com- pletion of the Human Genome Project and the emergence of Systems Biology, elucidation of complex intracellular signaling processes is being accomplished with cell-based assays.

RNA interference (RNAi) is rapidly emerging as a powerful tool for investigating the physiological function of through loss-of-function screens in stably transfected or primary cultures. Systems for delivering RNAs into cells for the purpose of silencing include and viral systems for the inducible expression of small interfering RNAs (siRNAs) in vivo. An efficient and widely applicable delivery method is lentiviral mediated introduction of expression cassettes that encode short hairpin RNAs (shRNAs).

The RNAi Consortium (TRC), based at the Broad Institute’s RNAi Platform, performs high throughput RNAi screening. The RNAi Platform screens have been automated using a unique integrated JANUS® Automated Workstation designed to meet the assay and biosafety requirements of the lentiviral infection protocol. Here we highlight the design and capabilities of this JANUS Workstation, and present the results of studies conducted to validate the performance of the automated system. Materials & Methods Reagents ATPlite® 1step and black ViewPlate®-384 TC are PerkinElmer products. Polybrene and Puromycin were obtained from Sigma-Aldrich (St. Louis, MO). Alamar Blue was purchased through Life Technologies (Carlsbad, CA).

Medium Change High throughput lentiviral RNAi gene knockdown screens are performed using a wide range of mammalian cells (primary or established cultured lines) grown in either 96-well or 384-well

microplates at 37ºC, 5% CO2. Throughout the course of the infection protocol the cell culture medium is changed as virus and various reagents are added or removed. Since the quality Two JANUS Mini Workstations and two PlateStaks as configured for the Broad Institute. of the data generated by the screen is dependent on a uni- form cell culture environment from well-to-well, the medium the presence of polybrene to enhance infection efficiency. removal and addition steps must be performed quickly without Duplicate sets of plates are prepared; one replicate is then disruption of cellular monolayers. exposed to puromycin and the other left untreated in order to determine infection efficiency. A typical workflow is out- Automation lined below: In designing the automated liquid handling system for - Day 0: Seed cells at the appropriate cell density in either mediated screening, biosafety issues also needed to be taken 96-well (100 µL per well) or 384-well (50 µL per well) into account. Since these screens are carried out in a BSL2+ sterile tissue culture treated plates. Incubate overnight. facility at the Broad Institute, the system had to have a small enough footprint and height to fit into a biological Day 1: Remove growth medium and add fresh medium safety cabinet. containing polybrene. • Infect cells with virus: incubate for 4 hours, In view of the above considerations, a system consisting of then change medium to remove virus two integrated JANUS Mini platforms capable of processing Day 2: Remove medium and add fresh medium. Puromycin plates in parallel was selected. A dual diving board PlateStak® is included in the medium added to one set of the located between the two JANUS Minis shuttles plates from duplicate plates for selection. one to the other. During the medium change procedure one of the workstations performs the medium removal step; the Day 6: Analyze infected cells plate is then moved to the second workstation where the fresh medium is added. The advantages of using a dual system Medium Change Protocol Validation to perform the medium change rather than using a single When developing an automated cell-based assay it is essential workstation are: to validate that the readout of the assay is not affected by 1. Greater throughput with two units running in parallel instrument-introduced artifacts. This is particularly important for lentiviral-mediated shRNA gene knockdown screens due 2. Less time for delicate cell types to be in the absence of to the number of medium changes in the procedure, and culture medium the long incubation time for the assay. Since the infection 3. No cross contamination of old and new medium, so tip efficiency is determined by comparing the cell number in changing is not required saving considerable cost of plus-puromycin vs. minus-puromycin treated samples, the consumables liquid-handling by the JANUS during the aspiration and dis- In addition, either of the JANUS Minis can also be used pensing steps of the medium changes should not disrupt the standalone for high precision viral infections. cell layer and introduce inaccuracy in the results. The medium change protocol was validated by measuring the Workflows precision of the cell number from well-to-well after performing RNAi screening is carried out by the Broad’s RNAi platform the sequence of steps in the infection protocol using an empty using lentiviral libraries as the vector to introduce shRNAs lentiviral vector, and at the end of the sequence measuring the into cells, along with the puromycin resistance gene for cell number using ATPlite 1step reagent. Intracellular ATP is a selection of infected cells. Cells are treated with virus in marker for cell number, since it is present in all metabolically active cells.

2 A batch of eight plates were seeded at 500 cells/well in 50 µL This demonstrates that proper culture and medium change of culture medium and incubated overnight at 37 ºC, of the cells over the six day processing protocol is main-

5% CO2. The ATPlite 1step assay was performed on three tained by the JANUS Workstation. of the plates as a control to measure the cell-seeding preci- sion. The series of three medium changes described above Figure 2 below shows the plate-to-plate variation in cell number in the screening protocol process flow were carried out on and demonstrates that over a multiple-plate automated protocol, the other five plates, and at the end on the six day process results are consistent across the five test plates. the ATPlite assay measured the well-to-well cell number. The results are shown below, and demonstrate that throughout the course of the protocol the well-to-well precision of the Media Change Repeatability cell number is not affected by the liquid handling. 250,000 230,000 Screening Assay Protocol: Infection Efficiency 210,000 190,000 The dual JANUS Mini system is routinely used in high 170,000 throughput lentiviral-mediated RNAi screens carried out by 150,000 the RNAi platform. The dual integrated system is used for 130,000 the medium changes, and in addition, one of the JANUS Signal ATPLite 110,000 Minis is used as a standalone system for the viral infection 90,000 step. Infections are performed in duplicate, and puromycin 70,000 50,000 added to one of the replicates to select for infected cells. Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Cell number is determined by measuring the fluorescent signal using the Alamar Blue assay. Infection efficiency is Figure 2. Plate-to-Plate repeatability of cell number after the 6-day culture protocol determined by measuring the number of cells in paired wells with and without exposure to puromycin. In the course of a Screening Assay: Infection Efficiency screen control plates containing known lentiviral markers are included to ensure the system is performing properly. Using the RNAeye software developed at the Broad Institute, the results of a control plate included in a screen run by the RNAi platform was analyzed. A scatter plot was generated Results & Discussion of the signal from each well comparing the puro (+) plate to Medium Change Protocol Validation puro (-) plate as shown in Figure 3 below. Wells are considered Figure 1 below shows that the well-to-well variation in cell infected if the signal ratio of puro (+)/puro (-) exceeds 0.25. number in the plates whose medium was changed by the Using these criteria, a histogram shown at the bottom of the JANUS Automated Workstation remained at or below the figure demonstrates the high efficiency of the infection. level established for the control plates.

Cell Seeding Validation 16.0%

14.0%

12.0%

10.0%

8.0%

6.0%

Well-to-Well CV Well-to-Well 4.0%

2.0%

0.0% 1 2 3 1 2 3 4 5 Control Plate Media Change Plates

Figure 1 Well-to-well precision of cell number. Cellular ATP levels were measured just after cell seeding for the 3 control plates and after the 6-day culture protocol for the 5 test plates. Figure 3. Scatter plot and histogram of puro (+) vs. puro (-) signal and histogram of the ratio puro (+)/puro (-).

3 The screening data can also be visualized and scored in plate Conclusions format as shown in Figure 4 below. An integrated liquid handling system consisting of two JANUS Mini Workstations and two PlateStaks was developed to automate high throughput lentiviral RNAi screening. The system is capable of performing all of the steps in the infection protocol. Integration software allows the two workstations to operate in parallel resulting in higher throughput, better handling conditions with sensitive cell types, as well as labor and cost savings. Figure 4. Plate Layout View for Raw and Scored Data

The wells in the plate map of the scored data shown on the Acknowledgements right which are marked with a black line in the center are The authors wish to thank the Broad Institute for allowing control wells that are either left empty as a quality check the use of the system for the validation studies, and for to ensure that there has not been cross contamination dur- sharing the control virus plate (CTR01) data. ing the pipetting steps, or they are control wells containing control medium only. Data can be scored by selecting the desired statistical normalized method to be applied, such as b-score, z-score, or robust z-score, in order to calculate gene scores from hairpin scores.

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