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Custom Pooled shRNA Libraries

Pooled lentiviral-based libraries containing heterogeneous mixtures of shRNA allow you to assay the effects of many thousands of pooled constructs in one experiment. By taking advantage of array-based oligonucleotide synthesis, we can readily make precisely defined large custom pooled libraries expressing many thousands of shRNAs. In approximately 2 months, we can produce a completely new, high-quality custom library focusing on any target set of genes. The library vector can also be easily customized, and the same library oligonucleotides can be cloned into more than one vector backbone (e.g. with an inducible and constitutive shRNA). It is a very flexible platform.

Unambiguous Sequenceable Barcodes

Screening readout of enrichment or depletion representation of particular shRNAs is greatly facilitated by the incorporation of easily sequenced barcodes in each shRNA construct. Direct amplification and sequencing of hairpins is problematic, and increase variability in the data since small sequence variations of the hairpin stem significantly affect the efficiency of next-generation sequencing (NGS). Separate shRNA-specific barcodes that are optimized for sequencing, on the other hand, enable unambiguous identification of each shRNA species with NGS.

Effective shRNA

In pooled lentiviral library construction, one key challenge is the design and production of quality oligonucleotides to provide the shRNA inserts for the libraries. Cellecta has carried out extensive internal studies to determine the best structural features (e.g. length, loop size, mismatches, etc.) and sequence targeting to use for pooled RNAi screens.

The results of this work, combined with published information regarding sequence preferences shown effective for particular targets, provide Cellecta with an optimized shRNA scaffold and its own in-house design algorithm to produce highly effective pooled shRNA libraries.

We can design libraries to target each transcript with as many shRNAs as desired. Given that shRNA can often have off-target effects due to its similarity with miRNA regulator sequences, we usually design shRNA libraries with 10 or more hairpins to each specific transcript. More than two-thirds of our optimized shRNAs knock down transcript levels by greater than 70%.

User Manuals

shRNA Libraries

  • Custom Pooled Lentiviral shRNA Libraries, v4 (PDF)
  • Pooled Lentiviral shRNA Library Screening Reference Manual, v2a (PDF)

CRISPR sgRNA Libraries

  • Custom and Premade Pooled Lentiviral CRISPR sgRNA Libraries, v2 (PDF)

Next-Gen Sequencing of Samples from Genetic Screens

Cellecta shRNA and CRISPR libraries are provided with a complete protocol and all sequencing information to enable researchers to perform high throughput genetics screens and analysis. However, we do also provide Next-Gen Sequencing (NGS) and analysis services for researchers running their own screens with our libraries. Just provide harvested cells for each time point or treatment condition (one sample), and we extract DNA, amplify, sequence, and assemble the data with some basic analysis.

You provide Cellecta with frozen cell pellets or tissue after screening…

…and Cellecta does the rest:

  • Extracts genomic DNA
  • Amplifies barcodes or sgRNA sequences
  • Performs NGS on the Illumina NextSeq or HiSeq
  • Enumerates shRNA or sgRNA counts from raw sequencing data

Please see the Next-Gen Sequencing and Analysis web page for additional information and how to order.

Custom CRISPR sgRNA Libraries

Custom shRNA Libraries