NGS Reagents for Pooled shRNA Libraries

High efficiency NGS readout of pooled shRNA library screen

After shRNA knockdown screens, the frequency of each integrated shRNA is assessed by next-generation sequencing (NGS) to determine which hairpins are enriched or depleted relative to the original libraries or control samples.

To streamline this process, Cellecta´s NGS Prep Kits for Pooled shRNA Libraries can be used to amplify, sequencing and accurately quantify the representation of shRNAs after running screens with Cellecta´s pooled lentiviral shRNA libraries.

The Kits contain all reagents necessary reagents, including highly specific PCR primers for efficient amplification of shRNA-cassettes as well as indexed primers to prepare sequence-ready libararies for multiplex NGS analysis.

NGS Prep Kits features

Optimized protocols and reagents enable PCR reactions with a gDNA input of as high as 50 µg per reaction, which significantly reduces the number of PCR reactions required
   ⇒ Cost and time savings by substantially fewer PCR reactions compared to conventional low gDNA-input methods

– No background due to the reduced number of PCR cycles required

– Kits contain Index Primers for multiplexing up to 12 samples

– Free Data Analysis Software included: the program demultiplexes, aligns, and scores Illumina platform sequence data

– Kits are also available for all Cellecta sgRNA-, Barcode-  and RNA-Seq Barcode Libraries.

Workflow

– gDNA isolation from samples of genetic screens

– First Round PCR: shRNA-cassettes are amplified with primers  that are highly specific to the shRNA library vector backbone

– Second Round PCR: Introducing of indexes and P5/P7 adapter to generate sequence-ready NGS libraries

– NGS on an Illumina sequencing platforms (e.g., NextSeq, NovaSeq, HiSeq, or MiSeq)

– Data analysis: the program demultiplexes indexed samples and aligns sgRNA reads from the Illumina NGS FASTQ files. The output is a spreadsheet with the counts of each sgRNA in each sample.

In addition to NGS Prep Kits, we also offer Cellecta’s next-generation sequencing and analysis services for Cellectas sgRNA–, Barcode-  and RNA-Seq Barcode Libraries as well as for GecKO and Brunello sgRNA libraries.

Simply provide cells or gDNA from your genetic screen, and we will deliver a spreadsheet enumerating the read counts for each of the  library elements as well as  comprehensive statistical NGS data analysis report.