human starr seq ori vector  (Addgene inc)

Journal: bioRxiv

Article Title: Challenges and considerations for reproducibility of STARR-seq assays

doi: 10.1101/2022.07.27.501795

Figure Lengend Snippet: (A-E): Schematic representation of a STARR-seq protocol highlighting all sequence information. Schematic of STARR-seq experimental protocol and illustrating complete sequence information is shown. (A) Insert preparation including addition of sequencing adapters and cloning arms to library fragments to facilitate sequencing and library cloning. (B) STARR-seq plasmid library preparation including vector linearization and cloning of library inserts into human STARR-seq vector followed by library amplification through transformation. (C) Preparation of ‘input’ sequencing library either directly from plasmid library or from DNA extracted from library-transfected host cells. This step adds on UMIs and index barcodes to the library prior to sequencing to sort for PCR duplicates and to sequence multiple libraries on the same sequencing lane (multiplexing). (D) cDNA library generation for STARR-seq screening includes transfection of the plasmid library into a host cell and reverse transcription of self-transcribed reporter transcripts. This step also involves adding UMIs for detecting and removing PCR duplicates. (E) Preparation of ‘output’ sequencing library involves adding sequencing barcode indexes to the screening library prior to sequencing to enable multiplexing. Both ‘input’ and ‘output’ libraries are pooled and sequenced in parallel for enhancer screening.

Article Snippet: Use human STARR-seq vector ADDgene #99296 as reported by Muerdter and colleagues .

Techniques: Sequencing, Clone Assay, Plasmid Preparation, Amplification, Transformation Assay, Transfection, Multiplexing, cDNA Library Assay