75N95019Q00166_Statement_of_Work.pdf

STATEMENT OF WORK (SOW)

Analysis of the genomic methylation status of hPSC and iPSC

GENERAL INFORMATION

Title of Project:

Analysis of the genomic methylation status of hPSC and iPSC

Statement of Need and Purpose:

The Stem Cell Translation Laboratory (STCL) in the National Center for Advancing Translational

Sciences (NCATS) at the National Institutes of Health (NIH) requires analysis of the genomic methylation status of human pluripotent stem cells (hPSC) and their differentiated progeny – trophectodermal cells, and time course analysis of the genomic methylation status and open chromatin regions during induced pluripotent stem cell (iPSC) differentiation to astrocytes.

The required services include:

• Methylation analysis methods: MeDIP Sequencing – using next-generation sequencing to determine the methylation status of selected genomic loci or genome-wide. Methylation is a prominent mechanism of silencing genes and creating a tissue-specific pattern of gene activity.

• ATAC seq: This assay provides genome-wide profiles of open and accessible regions of chromatin that are indicative of active regulatory regions. This assay will help provide mechanistic insight into gene regulation, and cellular response to compounds used to differentiate stem cells into astrocytes.

These services will be utilized by NCATS to study the epigenetic profiles of human pluripotent stem cells (hPSC) and their differentiated progeny – trophectodermal/trophoblast cells and astrocytes. The data obtained will be used to construct a high-resolution “map” of the genomic and epigenomic landscape that controls human pluripotency and trophectodermal, and neuroectodermal/astroglial, differentiation.

Background Information and Objective:

The SCTL has recently developed a protocol for differentiating hPSC into trophectoderm and into astrocytes. Trophectoderm is an extraembryonic cell layer of an embryo that gives rise to the placenta during development. Astrocytes are most numerous non-neuronal cells in the brain that play important role in normal brain development, synaptic function, neurodegenerative diseases, and various pathological conditions (e.g. opioid addiction).

Solicitation No. 75N95019Q00166

Derivation of human trophoblast cells from pluripotent stem cells is challenging, partly due to the difficulty in identifying markers that define cells as belonging to the trophoblast lineage. Criteria have recently been selected that are characteristic of primary first-trimester trophoblast: a set of protein markers, HLA class I profile, methylation of ELF5, and expression of microRNAs

(miRNAs) from the chromosome 19 miRNA cluster (C19MC). Recently, the extent of imprinted differentially methylated regions (DMRs) has been observed to be extremely similar between individual human tissues, with the exception of the placenta. This extra-embryonic tissue often adopts a different methylation profile compared to somatic tissues. Therefore, analyzing the methylation status in the trophectoderm cells that have been derived from hPSC is of paramount importance for the characterization of the cells.

Derivation of human astrocytes from a scalable source such as induced pluripotent stem cells

(iPSCs) is an attractive approach for disease modeling and drug discovery, however, currently available protocols are variable, inefficient, and lengthy (lasting up to several months) due to inability to efficiently and selectively induce gliogenesis over neurogenesis. Therefore, analyzing the methylation status together with assessment of genome wide accessible regions of chromatin will provide us with an insight into a role of epigenetic modulators in astrocytes differentiation.

Period of Performance:

The period of performance is twelve (12) weeks, including receipt of all deliverables. The 12-week period of performance will begin when the contractor receives the samples from the

SCTL. The contractor will likely, but is not guaranteed to, receive the samples within 30 days of order.

SCOPE OF WORK

General Requirements:

Independently and not as an agent of the Government, the contractor shall furnish all the necessary services, qualified personnel, material, equipment, and facilities, not otherwise provided by the Government as needed to perform the general and specific requirements below.

After receipt of the samples, the contractor will extract the nucleic acids from the cell pellets, will perform quality analysis, construct sequencing libraries, perform sequencing on an Illumina platform, perform data analysis, and biological interpretation. The contractor will provide the data to the SCTL via FTP or HTML download.

Specific Requirements:

The contractor will perform MeDIP Sequencing for two (2) sample sets and ATAC-seq for one

(1) sample set. The contractor will analyze and deliver the data to the SCTL.

MeDIP-Seq

In MeDIP-Seq, a highly specific antibody that recognizes 5-methylcytosine is used to immunoprecipitate sonicated genomic DNA, resulting in the enrichment of genomic regions that are methylated. Because 5-methylcytosine antibody binds only to methylated cytosines in the context of single-stranded DNA, the DNA must be denatured prior to immunoprecipitation. As a result of denaturation, the enriched DNA cannot be processed for Next-Gen sequencing using the typical sequencing library generation protocols, as these require adaptor ligation to double-stranded DNA. This problem is circumvented by ligating the Next-Gen sequencing adaptors to genomic DNA prior to the immunoprecipitation. Following MeDIP, the enriched regions can be directly amplified with Next-Gen sequencing compatible primers. Unique alignment of the sequence tags across the genome reveals the regions of DNA methylation.

Several methodologies exist that can detect DNA methylation on a genome-wide scale. Whole genome bisulfite sequencing is the most comprehensive, providing single base resolution across the entire genome. However, it requires sequencing of the entire genome, which is extremely expensive. Reduced Representation Bisulfite Sequencing (RRBS) also yields single base resolution, but it only interrogates 10% of all CpGs and it is more heavily biased toward

CpG-rich regions than any other technique. This is not necessarily an advantage as most CpG islands are unmethylated, and because there is growing interest and focus on regions with lower CpG density. MeDIP-Seq coverage is not as biased toward high CpG density and in theory can interrogate all regions across the genome. The expanded genomic coverage of

MeDIP-Seq compared to RRBS increases the likelihood of identifying differentially methylated regions in multi-sample studies.

ATAC-Seq

ATAC-Seq identifies accessible DNA regions by probing open chromatin with hyperactive mutant Tn5 transposase that inserts sequencing adapters into open regions of the genome. The mutant Tn5 transposase excises any sufficiently long DNA in a process called tagmentation: the simultaneous fragmentation and tagging of DNA performed by Tn5 transposase pre-loaded with sequencing adaptors. The tagged DNA fragments are then purified, amplified by PCR and sent for sequencing. Sequencing reads can then be used to infer regions of increased accessibility as well as to map regions of transcription-factor binding sites and nucleosome positions. The key element of the ATAC-seq procedure is the action of the transposase Tn5 on the genomic

DNA of the sample. Transposases are enzymes catalyzing the movement of transposons to other parts in the genome (5). While naturally ocurring transposases have a low level of activity, ATAC-seq employs a mutated hyperactive transposase.

Analysis Sample Size Experimental Layout of samples (subject to change)

Contractor Responsibilities

MeDIP-Seq 12 cell pellet samples

• H9 ESC, 3 replicates

• H9-derived trophectodermal cells, day 10, 3 replicates

• WA17 ESC, 3 replicates

• WA17-derived trophectodermal cells, day 10, 3 replicates

• Extract nucleic acids

• Perform QC of the nucleic acid samples

• Perform MeDIP using a 5-methyl-Cytosine monoclonal antibody

• Amplify the enriched DNA sample

• Perform Next-Gen sequencing

MeDIP-Seq 15 cell pellet samples

• Lonza iPSC, 3 replicates

• Lonza iPSC-derived astrocytes cells, day 7, 14, 21 and 30, each 3 replicates

• Extract nucleic acids

• Perform QC of the nucleic acid samples

• Perform MeDIP using a 5-methyl-Cytosine monoclonal antibody

• Amplify the enriched DNA sample

• Perform Next-Gen sequencing

ATAC-Seq 15 cell pellet samples

• Lonza iPSC, 3 replicates

• Lonza iPSC-derived astrocytes cells, day 7, 14, 21 and 30, each 3 replicates

• Extract nucleic acids

• Perform QC

• Perform transposase reaction

• Library amplification

• Perform Next-Gen sequencing

• Bioinformatic analysis

DELIVERABLES

The following deliverables are required for each analysis:

• Raw data (FASTQ) output files

• Aligned data (BAM) files

• Differential methylation analysis tables

• Figures & graphs – Includes coverage depth analysis, heatmaps & pie charts

All reports will be provided to the SCTL in electronic form – PDF, MS Excel sheets, MS Word, TXT files, PNG, TIFF figures, or other format based on the type of report. Raw sequencing data will be provided in the form of FASTQ files and will be furnished using as an FTP or HTML server download.

GOVERNMENT RESPONSIBILITIES

The SCTL will derive the cell pellet samples from the biological material. The cells will be pelleted in 1.5ml Eppendorf tubes in sufficient numbers: 2 to 5 million. The cell pellets will be flash frozen on dry ice and will be shipped to the contractor on dry ice. The samples will likely, but are not guaranteed to, be shipped to the contractor within 30 days of order.

OTHER CONSIDERATIONS

Data Rights:

All data generated as a result of all services will be the properties of the Stem Cell Translation

Laboratory, the National Center for Advancing Translational Sciences, and the National

Institutes of Health. The contractor shall have no rights to any data generated.

Publications and Publicity:

The datasets derived by the SCTL from these experiments will be part of scientific publications that will be made available to the public on the SCTL public-facing internet website. The contractor will not publish any result of the work.

Confidentiality of Information:

The contractor must keep the results of all work performed under the contract confidential.