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RNAi Core

Ralph Garippa (Core Facility Head)

Office phone

646-888-3339

Email(s)

garippar@mskcc.org

The RNAi Core Facility supports Memorial Sloan Kettering investigators with resources and tools related to RNA interference (RNAi) technologies, which can be used to study individual genes, dissect signaling pathways, and identify potential drug targets. We provide existing siRNA and shRNA libraries, customized shRNA reagents, and RNAi screening services.

Services

RNAi Ordering Services

The RNAi core provides the following reagents:

  • Stem-loop shRNA clones from The RNAi Consortium (TRC) libraries supplied by Sigma-Aldrich (TRC 1.0, 1.5 and 2.0). These include 100,000 clones against over 16,000 human and mouse genes, and appropriate lentivirus transduction controls supplied by Sigma-Aldrich.
  • Pooled siRNAs from two human siRNA libraries supplied by Thermo Fisher Scientific, siGENOME, and ON-TARGETplus. These include 18,174 pooled siRNAs against 19040 genes.

mirR30/miRE/CRISPIR libraries ready off the shelf
Library Gene# Subpool# shRNA/sgRNA# per gene Status
human druggable genome 2263 6 5 with control completed
human cancer metabolome 639 2 5 with control completed
human DNA damage repair 1825 5 5 with control completed
mouse DNA damage repair 310 6 10 completed
human kinome 526 3 5 with control completed
human epigenome 565 2 5 with control completed
human phosphatase 254 1 5 with control completed
human nuclear hormone receptor 49 1 5 with control completed
human oncogene (Vogelstein, ref1) 62 1 6 with control completed
human tumor suppressor (Vogelstein, ref1) 73 1 6 with control completed
mouse oncogene (Vogelstein, ref1) 62 1 6 with control completed
mouse tumor suppressor (Vogelstein, ref1) 73 1 6 with control completed
mouse Phagosome-proteosome 1385 10 6 completed
mouse drugged genome 446 3 5 with control completed
mouse DNA methylation 495 5 5 with control completed
mouse ion channelome 223 1 5 with control ongoing
human SAM library (ref2) 23430 1 3 with control under QC
human CRIPSR library (Brunello, ref3) 19114 1 4 with control under QC
Mouse CRISPR library (Brie, ref3) 19674 1 4 with control under QC
human GeCKO library v2 (ref4) 19050 2 6 with control completed
mouse GeCKO library v2 (ref4) 20611 2 6 with control completed
human GeCKO library v1 (ref5) 18080 1 2 to 4 completed

Completed: maxiprep’d, stocked, sequenced, QC’d
ref1: Bert Vogelstein et al, ” Cancer Genome Landscapes” Science 2013, 339: 1546-1558
ref2: Konermann et al, “Genome scale transcriptional activation by an engineered CRISPR-Cas9 complex” Nature 2015, 517:583-8
ref3: Doench et al, “Optimized shRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9” Nat Biotechnol 2016, 34:184-91
ref4: Neville Sanjana et al, “Improved vectors and genome-wide libraries for CRISPR screening” Nature Methods 2014, 11: 783-784
ref5: Ophir Shalem et al, “Genome-scale CRISPR-Cas9 knockout screening in human cells” Science 2014, 343: 84-87

Focused shRNA Pooled Libraries

Working in close collaboration with the laboratory of Scott W. Lowe, of the Sloan Kettering Institute’s Cancer Biology and Genetics Program, we provide investigators with various focused mir30 shRNA pooled libraries that target specific gene groups such as kinome, epigenome, drugged genome, DNA-damage-repair-response genes, and many others.

Custom miR-30 shRNA and high-potency miR-E shRNA

Upon receiving a list of genes of interest to an investigator, the RNAi Core designs shRNAs against these genes using algorithms developed by Dr. Lowe’s lab. It then constructs off-the-chip libraries, and makes recommendations for experimental optimization.

The choice of vector backbone, lentivirus or retrovirus delivery system, fluorescent markers, promoters, and selection markers can be provided in either a constitutive or inducible format. We clone individual shRNA and pool libraries.

CRISPR-cas9 systems, individual and screening libraries

In addition to Genome-scale GeCKO library, we provide custom order individual CRISPR or screening libraries.

RNAi Screening

The RNAi Core conducts RNAi-based screening of pooled shRNA libraries, arrayed focused shRNA libraries (using one individual shRNA per well), and siRNA libraries (using pooled siRNA against one gene per well).

Our shRNA pools are offered for positive or negative selection screen, usually analyzed by cell sorting through fluorescence reporters, followed by deep sequencing. The screening readouts for arrayed libraries may be cell viability (such as luminescence-based ATP measurement or fluorescence-based redox activity measurement) via a plate reader, or high-content fluorescent image quantification with an automated fluorescence microscope.

The RNAi screening service covers development and optimization, screen execution, data analysis, and candidate gene nomination with follow-up confirmation studies such as secondary screens and reversal of gene silencing by expressing cDNA. The initial consultation to evaluate the technical feasibility of the assay is free.

Compound Screening

We provide high content screening (HCS) using automated fluorescence microcospopy in 2D and 3D, high throughput screening (HTS) with assay development, cell-based and biochemical readouts, liquid handling and plate washing, and a ~350,000 small molecule compound library including FDA-approved drugs, known bioactive agents, diversity sets, kinase inhibitor subsets, natural products, and MSK internal chemistry compounds.