Attachment 1 Purchase Description.pdf

Version 11-30-2016-1 FAR Part 11—Describing Agency Needs

PURCHASE DESCRIPTION

1. Statement of Need and Purpose:

As the first results of single cell RNA sequencing dataset are about to publish there are increasing need for more experiments on sequencing single cell 3' and spatial gene expression using Visium product to continue the research and prepare for the next publication. The purpose of this acquisition is to prepare for these next experiments and reduce the interval between procurement and research schedule.

These kits are the key component to profile cell types and identify novel targets with single cell RNA-seq in marmoset central nervous system. The goal of this study is to establish a database system for marmoset brain cell type to reveal the correlation between specific cell types and stages of multiple sclerosis. Currently these are the only kits in the market that provides the specificity the study is designed for in a single cell level. Chromium Next GEM single cell 3' GEM, library & gel bead kit v3.1 and Visium Spatial Gene Expression Reagent Kit are designed for 16 reactions and there are total number of 32 samples to process, which requires these two kits to finish the project. These kits are ultra-sensitive to small population of cells. Required no previous knowledge of the sequence of our species. Unlike human and mice, there is no pre-designed primers panel for marmoset. These kits can capture the feature of the mRNA and barcode individual RNA molecule by unique identifier. Therefore, once the sequence is decoded, they will be able to track the molecule and cell types for any species including marmoset.

2. Background Information and Objective:

The National Institute of Neurological Disorders and Stroke (NINDS) is a part of the National Institutes of Health (NIH), conducting research into the causes, treatment, and prevention of neurological disorders and stroke. The Translational Neuroradiology Section is expanding its research from advanced MRI and histopathology to genomic and molecular field to elucidate the disease manifestations of multiple sclerosis. The chromium Next GEM single cell 3' GEM, library & gel bead kit v3.1, Chromium Next GEM Chip G Single Cell Kit, Visium Spatial Gene Expression Reagent Kit, Dual Index Kit TT Set A, and Visium Spatial Tissue Optimization Slide & Reagent Kit are required to unveil which cell types play critical roles in developing multiple sclerosis in animal model.

Generic Name of Product:

Chromium Next GEM single cell 3' GEM, library & gel bead kit v3.1, 16rxns

Chromium Next GEM Chip G Single Cell Kit, 16 rxns Visium Spatial Gene Expression Reagent Kit, 16rxns Dual Index Kit TT Set A, 96rxns Visium Spatial Tissue Optimization Slide & Reagent Kit, 4 slides

4. Purchase Description:

Trade Name: N/A Brand Name: Chromium Next GEM single cell 3' GEM, library & gel bead kit v3.1, 16rxns Maufacturer’s Name: 10X Genomics Model, type, catalog and other parts/numbers: Part number # 1000121

Trade Name: N/A Brand Name: Chromium Next GEM Chip G Single Cell Kit, 16 rxns Maufacturer’s Name: 10X Genomics Model, type, catalog and other parts/numbers: Part number # 1000127

Trade Name: N/A Brand Name: Visium Spatial Gene Expression Reagent Kit, 16rxns Maufacturer’s Name: 10X Genomics Model, type, catalog and other parts/numbers: Part number # 1000184

Brand Name: Dual Index Kit TT Set A, 96rxns

Model, type, catalog and other parts/numbers: Part number # 1000215

Brand Name: Visium Spatial Tissue Optimization Slide & Reagent Kit, 4 slides

Model, type, catalog and other parts/numbers: Part number # 1000193

5. Salient characteristics

1) Contract shall provide the NINDS with one Chromium Next GEM single cell 3' GEM, library & gel bead kit v3.1, 16rxns that include four major components:

Part1

Chromium Next GEM Single Cell 3ʹ Gel Bead Kit v3.1, 16 rxns PN-1000122 (store at −80°C) Chromium Single Cell 3ʹ v3.1 Gel Beads Single Cell 3ʹ v3.1

Item name Quantity Part number Gel Beads 2 2000164

Beads (#2000164) that coat with poly dT primer that enables capture mRNA inside the cells to make cDNA, at the same time provide two additional designed primers to capture sequences to enabling feature barcoding technology.

Part2

Chromium Next GEM Single Cell 3ʹ GEM Kit v3.1 16 rxns PN-1000123 (store at −20°C) Chromium Next GEM Single Cell 3ʹ GEM Kit v3.1

RT Reagent B 1 2000165 RT Enzyme C 1 2000085

Template Switch Oligo 1 3000228 Reducing Agent B 1 2000087

Cleanup Buffer 2 2000088 cDNA Primers 1 2000089

Amp Mix 1 2000047

Bead captured cell will then be lysed to release mRNA and captured by compatible cDNA primers (2000089) to match 16 nt 10X barcode and 12 nt unique molecular identitifier and 30 nt polydT sequence provided on the beads (2000059). RT reagent (2000165), RT enzyme C (2000085), template switch oligo (3000228), reducing agent B (2000087), and Amp mix (2000047) will be added to the reaction to reverse transcript mRNA to cDNA in a PCR machine.

Following reverse transcription, a reducing agent B (2000087), and cleanup buffer (2000088), and dynabeads (Part4 2000048) will be added to recover samples and following clean up steps.

Part3

Chromium Next GEM Single Cell 3ʹ Library Kit v3.1 16 rxns PN-1000157 (store at

Chromium Next GEM Single Cell 3ʹ Library Kit v3.1

Fragmentation Enzyme 1 2000090 Fragmentation Buffer 1 2000091

Ligation Buffer 1 2000092 DNA Ligase 1 220110

Adaptor Oligos 1 2000094 SI Primer 1 2000095

To prepared cleaned cDNA to a library that is compatible with next generation sequencing machine. A fragmentation enzyme (2000090), and fragmentation buffer (2000091) will be added to the solution completed by part 3. cDNA will then be broken to ~200bp pieces for downstream application. To ensure sequencing machine can capture the small cDNA pieces, a sdaptor oligos (2000094), and SI primer (2000095) will be added to the both end of the small cDNA in a buffer containing DNA ligase (220110) and amp mix (2000047). The reaction will be carried out in a PCR machine. And following clean up steps are as Part3 described, with dynabeads (Part4 2000048) to capture completed cDNA library.

Part4

Dynabeads™ MyOne™ SILANE PN-2000048 (store at 4°C)

Dynabeads MyOne

SILANE

1 2000048

The chromium Next GEM single cell 3' GEM, library & gel bead kit v3.1 will provide specific functions as below:

• The kit must identify and characterize rare cell types.

• The kit must analyze and understand cellular heterogeneity and how this contributes to your biological system.

• The kit must perform cellular phenotyping with single cell RNA-seq to identify novel targets, biomarkers, and cell types and states without the need for pre-selected targets.

• The kit must evaluate mRNA and cell surface protein expression profiles within the same cell.

• The kit must perform high-throughput and high-resolution functional genetic screens in tens of thousands of cells simultaneously.

• The kit must assess comprehensive gene expression phenotypes for individual CRISPR perturbations.

• The shelf life for the kit is 6 to 12 months.

2) Contract shall provide the NINDS with two Chromium Next GEM Chip G

Single Cell Kit, 16 rxns PN-1000127 that include three major components:

Chromium Next GEM Chip G Single Cell Kit (store at ambient temperature) Chromium Partitioning Oil

Partitioning Oil 2 2000190

Chromium Recovery Agent

Recovery Agent 2 220016

Chromium Next GEM Chip G & Gaskets

Chromium Next GEM Chip G

2 2000177

Gasket, 2-pack 1 3000072

Chromium Next GEM Chip G Single Cell Kit will provide specific functions as below:

• The kit is required to generate and recover up to 10,000 cells in each droplet

• The kit is required to generate droplets in 8 parallel channels to ensure minimize batch effect that might cloud biological reading.

• The kit is required to have a gasket cover that is aligned with the chip where microfluidic flow is generated to ensure optimized cell recover to study rare population.

3) Contract shall provide the NINDS with one Visium Spatial Gene

Expression Reagent Kit, 16rxns that include three major components:

Visium Spatial Gene Expression Reagent Kit, 16 rxns PN-1000186 (store at

Visium Spatial Gene Expression Reagent Kit

Permeabilization

Enzyme 1 2000214

RT Reagent 1 2000086 RT Enzyme D 1 2000216

Template Switch Oligo 2 3000228 Second Strand Primer 1 2000217

Second Strand Enzyme 1 2000218 Second Strand Reagent 1 2000219 cDNA Primers 1 2000089

A Permeabilization Enzyme (#2000214) is used to permeabilize the tissue sections on the slide. The polyadenylated mRNA released from the overlying cells is captured by the primers on the spots. RT Master Mix containing RT Reagent (#2000086), RT Enzyme D (2000216) is added to the permeabilized tissue sections. Incubation with the reagents produces spatially barcoded, full-length cDNA from polyadenylated mRNA on the slide. Second Strand Mix containing Template Switch Oligo (#3000228), Second Strand Primer (#2000217), Second Strand Enzyme (#2000218), Second Strand Reagent (#2000219), cDNA Primers (#2000089), and Amp Mix (#2000047) is added to the tissue sections on the slide to initiate second strand synthesis. This is followed by denaturation and transfer of the cDNA from each Capture Area to a corresponding tube for amplification and library construction. After transfer of cDNA from the slide, spatially barcoded, fulllength cDNA is amplified via PCR to generate sufficient mass for library construction.

Library Construction Kit, 16 rxns PN-1000190 (store at −20°C) Library Construction Kit

Fragmentation Buffer 1 2000091

Fragmentation Enzyme 1 2000090 Ligation Buffer 1 2000092

DNA Ligase 1 220110 Adaptor Oligos 1 2000094

Enzymatic fragmentation and size selection are used by mixing Fragmentation Buffer (#2000091), Fragmentation Enzyme (#2000090) together to optimize the cDNA amplicon size. P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) are added by mixing Ligation Buffer (#2000092), DNA Ligase (#220110), Adaptor Oligos (#2000094), and Amp Mix (#2000047) for End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina amplification.

Visium Spatial Gene Expression Slide Kit, 16 rxns PN-1000185 (store at ambient temperature) Spatial Gene Expression Slide Kit

Visium Spatial Gene

Expression Slide 4 2000233

Slide Seal 20 3000279 Slide Cassette 4 3000406 Slide Gasket 4 3000426

The Visium Spatial Gene Expression Slide (#2000233) includes 4 Capture Areas (6.5 x

6.5 mm), each defined by a fiducial frame (fiducial frame + Capture Area is 8 x 8 mm).

The Capture Area has ~5,000 gene expression spots, each spot with primers that include: Illumina TruSeq Read 1 (partial read 1 sequencing primer), 16 nt Spatial Barcode (all primers in a specific spot share the same Spatial Barcode), 12 nt unique molecular identifier (UMI), 30 nt poly(dT) sequence (captures poly-adenylated mRNA for cDNA synthesis). Slide Seal (#3000279) is used with Slide Cassette (#3000406) and Slide Gasket (#3000426) to hold Spatial Gene Expression Slide (#2000233) throughout the experiment to facilitate handling.

The Visium Spatial Gene Expression Reagent Kit, 16rxns will provide specific functions as below:

• The kit must identify and characterize rare cell types.

• The kit must analyze and understand cellular heterogeneity and how this contributes to your biological system.

• The kit must perform cellular phenotyping with single cell RNA-seq to identify novel targets, biomarkers, and cell types and states without the need for pre-selected targets.

• The kit must evaluate the expression profiles of total mRNA in intact tissue sections and maps the location where gene activity is occurring

4) Contract shall provide the NINDS with one Dual Index Kit TT Set A, 96rxns that include one major components:

Dual Index Kit TT Set A, 96 rxns PN-1000215 (store at −20°C) Dual Index Kit TT Set A

Dual Index Kit TT Set A 1 3000431

Dual Index Kit TT Set A, 96rxns will provide specific functions as below:

• The kit is required to be able to de-multiplex multiple samples in the same run of RNA-sequencing pipeline.

• The kit must be able to add P5, P7, i7 and i5 sample indexes at the same time

5) Contract shall provide the NINDS with one Visium Spatial Tissue

Optimization Slide & Reagent Kit, 4 slides PN-1000193 that include two major components:

Visium Spatial Tissue Optimization Reagent Kit, 4 slides PN-1000192 (store at

Visium Spatial Tissue Optimization Reagent Kit

RT Reagent C 1 2000215 RT Emzyme D 1 2000216 Template Switch Oligo 2 3000228 Reducing Agent B 1 2000087 Permeabilization Enzyme

2 2000214

Tissue sections are permeabilized for varying amounts of time using Permeabilization Enzyme (#2000214). Each Capture Area captures polyadenylated mRNA from the attached tissue section. A Master Mix containing reverse transcription (RT) reagents including RT Reagent C (#2000215), RT Emzyme D (#2000216), Template Switch Oligo (#3000228), Reducing Agent B (#2000087) and fluorescently labeled nucleotides is added on top of the tissue sections, #resulting in fluorescently labeled cDNA.

Visium Spatial Tissue Optimization Slide Kit PN-1000191 (store at ambient temperature) Visium Spatial Tissue Optimization Slide Kit

Visium Spatial Tissue Optimization Slide

4 3000394

Slide Seals 20 3000279 Slide Cassette 4 3000406 Slide Gasket 4 3000426 Tissue Removal Buffer 1 2000221 Tissue Removal Enzyme

1 3000387

Each Capture Area on the Visium Spatial Tissue Optimization Slide (#3000394) is defined by an etched frame. Each Capture Area contains oligonucleotides for mRNA capture. Each probe has poly(dT) primers that enables the production of cDNA from polyadenylated mRNA. These probes do not contain a spatial barcode. There are eight Capture Areas per slide. Seven of these are used for tissue and one is used for a positive RNA control. Only one tissue type should be tested per slide. Tissue is enzymatically removed using Tissue Removal Buffer (#2000221) and Tissue Removal Enzyme (#3000387) leaving behind fluorescent cDNA covalently linked to oligonucleotides on the Visium Spatial Tissue Optimization slide. Slide Seal (#3000279) is used with Slide Cassette (#3000406) and Slide Gasket (#3000426) to hold the Visium Spatial Tissue Optimization Slide (#3000394) throughout the experiment to facilitate handling.

Visium Spatial Tissue Optimization Reagent Kit will provide specific functions as below:

• The kit must be able to hold 8 cryosectioned per slide.

• The kit must allow tissue sections to be fixed with methanol and stained with hematoxylin and eosin (H&E).

• The kit must allow stained tissue sections to be imaged and aligned for comparison with final fluorescent images

• The kit must be able to have a fine tuned enzymatic reaction to facilitate condition priming.

6. Quantity: 1 Chromium Next GEM single cell 3' GEM, library & gel bead kits v3.1

16 rxns 2 Chromium Next GEM Chip G Single Cell Kit, 16 rxns 1 Visium Spatial Gene Expression Reagent Kit, 16rxns 1 Dual Index Kit TT Set A, 96rxns 1 Visium Spatial Tissue Optimization Slide & Reagent Kit, 4 slides

7. Delivery Date: The contractor shall deliver all kits between the hours of 8:00am and 5:00 pm, Bethesda, MD local prevailing time at 10 Center Drive, Room 5C103, MSC 1400, Bethesda, MD 20892. The kit will be delivered in ice, dry ice, and room temperature (conditions are indicated above) to maintain the temperature. Delivery is required within 30 days after award.