microTRE Screening Libraries
(Pooled Libraries #201012, #201013, #201014, #1000000220)

• Purpose:

  A massively parallel reporter assay (MPRA) library where barcode expression is dependent upon engagement of cellular transcription factors to a series of engineered, short, artificial transcriptional response elements (TREs) encoded alongside variable motifs and minimal promoters.

• Vector Backbone:

  Donor_eGP2AP_RC (Plate #133784)

• Depositing Labs: Justin English
• Publication: Zahm et al. bioRxiv. 2023 May 11:2023.05.11.539703. doi: 10.1101/2023.05.11.539703. Preprint.

Library Details

• Species: Synthetic, but based on human and mouse
• Synthetic promoters: 6318
• Unique barcodes: Each of the three pooled libraries contains approximately 1.2 × 10⁶ unique barcodes mapping to the TRE-barcode dictionary.
• Insert size range: 59–148 bp

Library Shipping

This library is delivered as suspended DNA in a microcentrifuge tube on blue ice. The tube's contents will not necessarily be frozen. For best results, minimize freeze/thaws.

• Volume: ∼20 µL
• Concentration: 50 ng/µL

Resource Information

• Protocols:
  • Pooled Library Preparation (DOCX, 13 KB)
  • PCR Protocol (DOCX, 14KB)
• Depositor Data:
  • Representative NGS Results (PNG, 179 KB)
  • Representative Sequence for minTK Library (GB, 16 KB)
  • Representative Sequence for minPromega Library (GB, 16 KB)
  • Representative Sequence for minCMV Library (GB, 17 KB)

Terms and Licenses

Academic/Nonprofit Terms:

• UBMTA
• Luciferase Limited Use Label License

Industry Terms:

• Not Available to Industry

Trademarks:

• Zeocin® is an InvivoGen trademark.

Depositor Comments

Figure 1. TRE-MPRA overview. A) Transcription factor binding motifs (TFBMs) included in the TRE-MPRA library were derived from the HT-SELEX dataset of Jolma et al., 2013. Candidate synthetic promoters were designed by combining homotypic TFBMs and one of three minimal promoters in multiple configurations. Commercially available TREs, as well as negative control promoters (‘pseudo’) were also included in the TRE-MPRA library. B) Four copies of a given TFBM were oriented on alternating sides of the DNA double helix using sets of random nucleotides and rotated along the helix relative to the minimal promoter (TRE unit). TRE units combined with minimal promoters (‘promoters’) regulate the expression of a Luc2 CDS containing 24 nucleotide barcodes in the 3’ UTR. Barcodes were mapped to associated TRE units using next-generation sequencing (NGS) during library preparation. C) Barcoded RNAs were extracted from transfected cells and sequenced via NGS alongside the input TRE-MPRA plasmid libraries. Resulting barcode counts were analyzed via MPRAnalyze (Ashuach et al., 2019) to produce estimates of promoter transcription rates and to compare activities between treatment with fetal bovine serum (FBS) or forskolin.

The Pooled NGS data provided by the depositor is from representative preps, and library composition changes very little between lots. NGS sequencing is required for the analysis pipeline, and datasets provided from the depositor can be used for comparative reference.

The necessary algorithms and barcode dictionary spreadsheet can be found on GitHub .

Individual dual-luciferase plasmids, pGL4.R_TRE_minTK (211516), pGL4.R_TRE_minPro (211517), and pGL4.R_TRE_minCMV (211518), can be used to clone and validate individual TREs from the corresponding library.