Statement of Work.doc

STATEMENT OF WORK (SERVICES)

(SOW)

GENERAL INFORMATION

Title of Project:

Mouse Whole Geneome Bisulfate Sequencing, Bulk RNA sequencing in the support of immune cell gene regulation, and Single Cell (sc) RNA sequencing in the support of Alzheimer’s disease related dementias research.

Statement of Need and Purpose:

We have a need to hire a contractor to perform the following:

· Whole Geneome Bisulfate Sequencing (WGBS) in mouse samples to examine the development of immune senescence in aging.

· Sequencing of bulk 1)RNA-seq, 2)ATACseq and 3) ChIPseq libraries are required to generate knowledge of the gene expression and transcriptional regulation both at the chromatin and chromosomal level. We require high throughput sequencing performed on a NextSeq to acheive the correct level of inquiry depth.

· The proposed sc RNA-seq experiment libraries are designed to evaluate changes in microglial composition and immune cell subsets in the brain, especially with regard to expression of inflammation-related genes, speciafically NFKB.

Background Information and Objective:

The analysis of DNA methylation state of heterochromatin in B, CD4+T and myeloid cells is best done by WGBS. WGBS is an especially complex form of whole genome sequencing because many cytosine associated with CG pairs get converted to thymines, leading to imbalance of C/G versus A/T ratios in the genome. Because of lack of experience within the NIA/IRP in this form of sequencing, this project is best done with an outside source.

The proposed bulk RNA-seq, ATACseq and ChIPseq experiments are designed to evaluate changes in in chromatin structure and gene expression in developing pro- and pre-B cells in the bone marrow in genetically mutated strains of mice, and to evaluate heterogeneity of activation responses in B and T lymphocytes, especially with regard to NF-kB-dependent gene expression and function of different subunits of NF-kB accessed via specific genetic mutations.

The proposed sc RNA-seq experiments are designed to evaluate a mouse model of Parkinson’s disease in which the disease time course can be followed temporally. These experiments will be carried in WT C57/Bl6 mice, in mice that lack specific immune cells, and in mice here the transcription factor NF-kB will be specifically manipulated in microglia. The sc libraries generated and sequenced here will provide a great deal of insight as to the function of NFKB in dementia.

Period of Performance:

The period of performance is anticipated to be 9/15/2020 to 8/30/2021.

SCOPE OF WORK

General Requirements:

The contractor shall generate WGBS libraries and sequence them.

The contractor shall sequence the bulk libraries provided by the government to obtain specified sequencing reads per sample on Illumina Novaseq machine. The contractor shall then provide FASTQ files as a resultant read out of the libraries provided.

The contractor shall sequence the single cell libraries provided by the government to obtain specified sequencing reads per sample on Illumina Novaseq machine. The contractor shall then provide FASTQ files as a resultant read out of the libraries provided.

Specific Requirements:

The contractor shall generate WGBS mouse libraries from 58 samples provided by the government. These 58 libraries will then be subsequently sequenced on an Illumina platform to 100Gb data per sample. The contractor shall then provide FASTQ files as a resultant read out of the libraries provided.

Illumina NextSeq instrument shall be utilized to sequence prepared bulk 1)RNA-seq, 2)ATACseq and 3) ChIPseq libraries. We are requsting the depth per library to be 400 million reads per run for RNAseq, ChIPseq libraries at 75 cycle sequencing and 130 million reads per library for ATACseq samples at 2X75 bp cycles. Once the libraries are sequenced, both the run details and the FATQ files will be delivered. To maintain uniformity with past experiments that is essential for rigorous data interpretation we need to use the same facility for future studies.

Illumina NovaSeq instrument shall be utilized to sequence prepared single cell RNA (scRNA) libraries. We are requsting the depth to be 500 million reads per sample for 3’ libraries and 200 million reads per sample for 5’ libraries in combination with 50 million reads for VDJ libraries. Once the libraries are sequenced, both the run details and the FATQ files will be delivered. To maintain uniformity with past experiments that is essential for rigorous data interpretation we need to use the same facility for future studies.

GOVERNMENT RESPONSIBILITIES

The Government will provide or generate the following and deliver them to the Facility Manager at the JHU Deep Sequencing and Microarray Core:

· 58 mouse samples

· The bulk libraries

· The scRNA libraries

DELIVERY OR DELIVERABLES

The contractor shall provide the fastq files via account access and on external hard desk.

Version 11-30-2016-1

FAR Part 11—Describing Agency Needs