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.
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.
|Library||Gene#||Subpool#||shRNA/sgRNA# per gene||Status|
|human druggable genome||2263||6||5 with control||completed|
|human drugged genome||729||2||5 with control||Summer 2015|
|mouse drugged genome||446||3||5 with control||Summer 2015|
|human cancer metabolome||639||2||5 with control||completed|
|human DNA damage repair||2144||6||5 with control||Summer 2015|
|mouse DNA damage repair||310||6||10||completed|
|human kinome||526||3||5 with control||Summer 2015|
|human epigenome||565||2||5 with control||completed|
|human phosphatase||254||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|
|human GeCKO library v2 (ref2)||19050||2||6 with control||completed|
|human GeCKO library v1 (ref3)||18080||1||2 to 4||completed|
|mouse GeCKO library v2 (ref2)||20611||2||6 with control||completed|
Completed: maxiprep’d, stocked, sequenced, QC’d
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.
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.
In addition to Genome-scale GeCKO library, we provide custom order individual CRISPR or screening libraries.
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.
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.