SOW.pdf

Statement of Work (SOW) Project: ATAC-Seq

GENERAL INFORMATION

1.0 Background:

• Transcriptional reprogramming is a key component of disease resistance.

MLA (mildew locus a) as an immune receptor that interacts with several transcription factors (TFs) represents a model to explore the regulation of nucleotide binding, leucine rich repeat (NLR) receptors during defense.

Previous work presented evidence that pointed to an interconnection between MLA-signaling and transcriptional reprogramming: first, two novel TFs were validated as interactors of MLA; second, the differentially resistant co-expressed interactome is associated with Mla through expression quantitative trait loci (eQTL) associations. Using time-series RNA-Seq data, a gene regulatory network (GRN) of barley immunity was reconstructed. Topological properties and gene ontology (GO) analysis were used to characterize the GRN, highlighting a hierarchical TF-structure of eight levels. Functional validation of these transcriptional relationships would reveal novel regulation mechanisms associated with the immune receptor. Experimental approaches to validate these TF-target associations include ATAC-Seq, which examines the accessible chromatin regions to elucidate the epigenetic mechanisms at work, and functional TF-target associations through virus-induced gene silencing (VIGS) and viral overexpression (VOX) (Lee et al., 2012). The validated TFs can be further studied for their effect in disease resistance and its maintenance when challenged with different powdery mildew strains.

ATAC-Seq is key to understanding the mechanisms of how the TFs in the GRN are activated and how this process is affected by MLA. This in turn will allow the engineering of NLR proteins with breeding purposes.

2.0 Scope of Work:

• A gene regulatory network (GRN) has provided a list of transcription factor (TF) candidates for validation of their role in disease resistance of barley to powdery mildew. USDA, Agricultural Research Service (ARS), Midwest Area (MWA), Corn Insect and Crop Genetics Research Unit in Ames, IA is interested in using ATAC-Seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) to test the chromatin accessibility and gene regulation of these TFs in the gene promoters of two barley lines: CI 16151 (Mla6), which is resistant to the powdery mildew fungus, and it’s derived susceptible mutant, mla6-m18982.

• The vendor will receive 36 frozen samples total = 2 genotypes (CI 16151, mla6-m18982 mutant) x 6 timepoints [0, 16, 20, 24, 32, 48 hours after inoculation (HAI)] x 3 biological replications.

• ATAC-Seq analysis will take into account:

o 36 libraries total o 2x150bp, Paired End reads (>50M reads/sample), single index o Quality check, alignment to reference barley genome, identification of accessible regions (peak calling).

CONTRACTOR REQUIREMENTS

1.0 Technical Requirements/Tasks:

• Contractor’s personnel must have knowledge of nuclei isolation and ATAC Sequencing from frozen plant (barley) tissue, not fixed tissue. Required ATAC- Seq service requirements include the following:

o ATAC-sequencing 36 libraries total = 2 genotypes (CI 16151, mla6-m18982 mutant) x 6 timepoints [0, 16, 20, 24, 32, 48 hours after inoculation (HAI)] x 3 biological replications.

Nuclei extraction and tagmentation-based library construction from frozen barley tissue.

2x150bp, Paired End reads (>50M reads/sample), single index, ATAC- Seq o Data Analysis Quality check, alignment to reference barley genome (Morex V2/V3), identification of accessible regions (peak calling).

• Contractor should consult with USDA-ARS client and provide expert bioinformatic analysis of differential regions of chromatin accessibility between samples.

2.0 Government Furnished:

• Government will furnish flash frozen 10 cm 1st leaves of barley (500 mg per sample).

5.0 Deliverables / Schedule:

Item No.

Deliverable

Objective

Due

1 Nuclei extraction and tagmentation-based library construction

Verify high quality libraries 1 month after contract award

2 ATAC-Sequencing

>50 million raw reads per mark on average. ≥80% of bases with quality scores of Q30.

2 months after contract award

3 Report Required to compare the samples and find genomic regions of interest.

2.5 months after contract award

All results to be provided to Technical Point of Contact listed below.

6.0 Travel: N/A

7.0 Contractor’s Key Personnel:

• Contractor’s personnel must have knowledge in ATAC Sequencing from frozen plant (barley) tissue. The contractor should offer expertise in nuclei extraction and tagmentation-based library construction from plant (barley) tissue.

8.0 Security Requirements: N/A

9.0 Data Rights: All material and data are the sole property of USDA-ARS Project 5030-21000-067-00D.

10.0 Section 508 – Electronic and Information Technology Standards: N/A

11.0 Technical Point of Contact/Location Monitor: Gregory Fuerst 515-294-2893 gregory.fuerst@usda.gov