Genomics Resources Core Facility

This protocol is optimized for cDNA synthesis from 1– 1,000 intact cells or ultra-low input amounts of total RNA. Due to the sensitivity of the protocol, the input material (total RNA or cells) needs to be collected and purified under clean-room conditions to avoid contamination. The whole process of SMART cDNA Synthesis should be carried out in a PCR Clean Work Station under clean-room conditions.

The Single Cell 5' protocols offer comprehensive, scalable solutions for measuring immune repertoire information and gene expression from the same cell. Profile fulllength (5' UTR to constant region), paired T-cell receptor (TCR), or B-cell receptor (BCR) transcripts from 500-10,000 individual cells per sample. A pool of ~750,000 barcodes are sampled separately to index each cell’s transcriptome. It is done by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA share a common 10x Barcode. Libraries are generated and sequenced and 10x Barcodes are used to associate individual reads back to the individual partitions. This document outlines the protocol to generate a T-cell library and/or a B-cell library, and/or a 5' Gene Expression library from amplified cDNA from the same cells.

• This service is only available for internal WCMC researchers.
• We accept the samples for QC including total RNA, cRNA, microRNA, genomic DNA, single stranded cDNA, double stranded cDNA and PCR product.
• Samples should be diluted with molecular biology grade water (RNase-free and Nuclease-free).
• Samples are tested for quality assurance on the Agilent 2100 Bioanalyzer and quantitated on the Nanodrop Spectrophotometer or Invitrogen Qubit Fluorometer as neccessary.
• Users must submit at least 3 µL of sample in a 1.5 mL microfuge tube.
• It is the user's responsibility to dilute the samples to the proper concentration range.

• RNA Quality Control.
• Sample submission: 3 ul (>50 ng/ul)

• RNA Quality Control.
• Sample submission: 3 µl (1-5 ng /µl).

The assay is for preparation of target- enriched, strand- specific RNA sequencing libraries from either high- quality RNA samples or formalin- fixed paraffin- embedded (FFPE) total RNA samples, without initial mRNA purification. This protocol is specifically optimized to enrich targeted regions of the transcriptome from repetitive sequences and sequences unrelated to the research focus prior to Illumina paired- end multiplexed sequencing.

Input total RNA: 100 - 200ng.

Genomic DNA ScreenTape:

For the analysis of genomic DNA from 200 to > 60,000 bp.

For the analysis of total RNA from 25 to 500ng/µL.

Sizing range: 35 - 1000 bp.

Quantitative Range: 0.1 - 50 ng/µL

High Sensitivity D1000 Reagents:

For the analysis of DNA from 35 to 1000 bp. 

High Sensitivity RNA ScreenTape:

For the analysis of total RNA from 500 to 10,000 pg/µL. 

The Archer FusionPlex ALK, RET, ROS1 v2 kit is a targeted sequencing assay to simultaneously detect and identify fusions and mutations of human ALK, RET and ROS1 genes. The kit is powered by Archer’s proprietary Anchored Multiplex PCR (AMP™) target enrichment chemistry to detect fusions of all genes in a single sequencing assay, even without prior knowledge of fusion partners or breakpoints.The kit also detects select insertions and point mutations in ALK and RET, including those reported in cell-based assays to convey crizotinib resistance

The Archer FusionPlex Solid Tumor Kit is a robust targeted sequencing assay to simultaneously detect and identify Fusions and other Mutations associated with over 50 genes linked to various carcinomas. Also included in the kit are primers that target known sarcoma- and hematological malignancy-associated Fusions as well as prominent BRAF and PDGFA Mutations.

The power of the FusionPlex Solid Tumor Kit lies in Archer’s proprietary Anchored Multiplex PCR™-based enrichment. This chemistry enables detection of all Fusions associated with the genes in this kit in a single sequencing assay, even without prior knowledge of Fusion partners or breakpoints.

It works with all clinical sample types, including FFPE.

Immunoverse BCR kits amplify B cell receptor heavy (IGH) and light (kappa/lambda) chains for sequencing and analysis. Primers are designed to identify isotypes and subclasses in the Fc region and somatic hypermutations within the variable regions. Sample inputs range from 25ng to 6μg.

The Archer Immunoverse TCR alpha/delta panel is a target-enrichment assay that can be used to create libraries for next generation sequencing of TCR alpha/delta chains. Libraries are created by using the Immunoverse alpha/delta assay in conjunction with the Archer Immunoverse TCR reagents and Archer MBC Adapters.

- Input nucleic acid (Total RNA) in EDTA-free buffer or Ultra-Pure Water is the optimal starting template for Archer AMP Library Preparation. Do not use EDTA-containing buffers exceeding 1mM.

- Use the maximum allowable input mass (ng) whenever possible.

- The minimum recommended input mass for Archer Immunoverse TCR assays is dependent on experiment type, tissue type and tissue quality: o 20ng of RNA for screening of high abundance clones from high-quality PBMC samples o 400ng of RNA for deep sequencing applications from high-quality, highcomplexity PBMC samples o 400ng of RNA from fresh-frozen tissue samples o >400ng for FFPE input for TIL analysis

- The rarefaction analysis below derived from varying amounts of high-quality input from a DNAse-treated PBL sample illustrates the relationship between input amounts, sequencing depths and clonotype count.

-  If using FFPE sample types, we recommend extracting TNA using Promega ReliaPrep™, Agencourt® FormaPure® or Promega Maxwell® RSC RNA FFPE Kit with the modifications to the published manufacturer protocol.

- It is recommended to use the Illumina® MiSeq® 600-cycle kit for the Immunoverse TCR alpha/delta kit.

The Archer Immunoverse TCR beta/gamma panel is a target-enrichment assay that can be used to create libraries for next generation sequencing of TCR beta/gamma chains. Libraries are created by using the Immunoverse beta/gamma assay in conjunction with the Archer Immunoverse TCR reagents and Archer MBC Adapters.

- Input nucleic acid (Total RNA) in EDTA-free buffer or Ultra-Pure Water is the optimal starting template for Archer AMP Library Preparation. Do not use EDTA-containing buffers exceeding 1mM.

- Use the maximum allowable input mass (ng) whenever possible.

- The minimum recommended input mass for Archer Immunoverse TCR assays is dependent on experiment type, tissue type and tissue quality: o 20ng of RNA for screening of high abundance clones from high-quality PBMC samples o 400ng of RNA for deep sequencing applications from high-quality, highcomplexity PBMC samples o 400ng of RNA from fresh-frozen tissue samples o >400ng for FFPE input for TIL analysis

- The rarefaction analysis below derived from varying amounts of high-quality input from a DNAse-treated PBL sample illustrates the relationship between input amounts, sequencing depths and clonotype count.

-  If using FFPE sample types, we recommend extracting TNA using Promega ReliaPrep™, Agencourt® FormaPure® or Promega Maxwell® RSC RNA FFPE Kit with the modifications to the published manufacturer protocol.

- It is recommended to use the Illumina® MiSeq® 600-cycle kit for the Immunoverse TCR alpha/delta kit.

Partial library prep. - if you submit tagmented DNA.

The 10x Genomics 3' CellPlex Kit provides a species agnostic sample multiplexing solution through the use of a set of 12 Feature Barcode oligonucleotides each conjugated to a lipid. Individual cells or nuclei samples can be labeled with a Cell Multiplexing Oligo (CMO) and then pooled together prior to loading onto a 10x
Genomics chip. The Feature Barcode molecules can be directly captured by the oligos present on the Gel Beads inside a GEM during GEM-RT and subsequently
amplified (see Stepwise Objectives for assay scheme specifics). The amplified DNA generated from the Feature Barcode molecules can be used for Cell Multiplexing
Library Construction. Upon sequencing and processing the data through Cell Ranger, pooled samples can be bioinformatically demultiplexed and analyzed as individual samples, with identified cell multiplets excluded.

Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for Cell Multiplexing.

The labeling cells/nuclei steps are performed in the user's lab using the kit 

3' CellPlex Kit Set A, 48 rxns PN-1000261 

Then bring the labeled cells or nuclei ready to the core lab for cell counting and further processing.

Highlights
1. Sensitive and Unbiased Characterization of
Transcriptional Signatures
Highly sensitive and reproducible gene detection in single
cells
2. Comprehensive Single-Cell RNA Sequencing Workflow
Fully supported workflow developed in collaboration by
technology innovators
3. Powerful Next-Generation Sequencing Integrated with
Simple Data Analysis
Proven Illumina sequencing combined with streamlined, userfriendly
analysis software

The Illumina DNA Prep protocol uses tagmentation, and enzymatic reaction, to fragment DNA and add adapter sequences and is compatible with DNA inputs of 1–500 ng or higher. For human DNA samples and other large complex genomes, the recommended minimum DNA input is 100–500 ng. For small genomes (eg microbial), the DNA input amount can be reduced to as low as 1 ng (modifying the PCR cycling
conditions accordingly). Assess DNA purity to make sure that the initial DNA sample does not contain > 1 mM EDTA and is free of organic contaminants, such as phenol and ethanol. These substances can interfere with the tagmentation reaction and result in assay failure.

upload sequencing data to GEO

Set up indexing PCR, pool, purification and QC of PCR products before sequencing. Charge $5/well (AOI).

llumina Total RNA Prep with Ribo-Zero Plus is based on TruSeq ligation technology. This ligation method results in high coverage uniformity, precise strand information, and robust and reliable library preparation even from degraded samples. 

The kit supports a broad range of RNA inputs, from 1 ng to 1000 ng. This input range is applicable to high-quality RNA. We recommend 10 ng input for best results and for FFPE or degraded samples.

It’s compatible with variable sample types, including formalin-fixed paraffin-embedded (FFPE) and other low-quality samples. The included Ribo-Zero Plus removes abundant RNA from multiple species, including human, mouse, rat, bacteria, and epidemiology samples.

The QuantSeq FWD Kit is an all-in-one library preparation protocol designed to generate Illumina compatible libraries of sequences close to the 3’ end of polyadenylated RNA.

The kit is ideal for gene expression counting as NGS reads are generated towards the poly(A) tail. To get the exact 3’ transcript end longer reads are necessary.

QuantSeq FWD Kits are delivered with several indexing options:

• Unique Dual Indexing with the Lexogen UDI 12 nt Sets (Cat. 113.96, 129-131.96, 114.96, 115.384) with up to 384 Unique Dual Indices.
• Single Indexing with the Lexogen i7 6 nt Index Sets (Cat. No. 015.24/96/2×96)
• Combinatorial Dual Indexing with the Lexogen i7 6 nt Index Set and i5 6 nt Dual Indexing Add-on Kit (015.384)

Output: 1 M reads / run.

You can perform a single read 50 cycles run.

This is a combined protocol for using NEBNext Ultra II Directional RNA Library Prep Kit and Twist Exome Kit #2.

Input total RNA: <100ng

30Gb / 100M reads / run.

40Gb / 400M reads / run.

80Gb / 400M reads / run.

120Gb / 400M reads / run.

100Gb / 1Billion reads

200Gb / 1 Billion reads

300 Gb / 1 Billion reads

50 GB / 1 Billion reads.

PCR amplicons as the input. The input amount: 10ng - 1ug based on which sample preparation protocol is used.

A poly-A based protocol and it contains the strand information. , it requires 25 - 100 ng of starting total RNA in 10-30 µl volume with an RIN value great than 7.8.

Procedure:

This sample preparation protocol that includes rRNA-depletion offers strand information on RNA transcript; Library capture of both coding RNA, as well as multiple forms of non-coding RNA; Degraded RNA can be used with minor adjustments to fragmentation procedures

Starts with total RNA.

The Single Cell 5' protocols offer comprehensive, scalable solutions for measuring immune repertoire information and gene expression from the same cell. Profile fulllength (5' UTR to constant region), paired T-cell receptor (TCR), or B-cell receptor (BCR) transcripts from 500-10,000 individual cells per sample. A pool of ~750,000 barcodes are sampled separately to index each cell’s transcriptome. It is done by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA share a common 10x Barcode. Libraries are generated and sequenced and 10x Barcodes are used to associate individual reads back to the individual partitions. This document outlines the protocol to generate a T-cell library and/or a B-cell library, and/or a 5' Gene Expression library from amplified cDNA from the same cells.

The 10x Genomics 3' CellPlex Kit provides a species agnostic sample multiplexing solution through the use of a set of 12 Feature Barcode oligonucleotides each conjugated to a lipid. Individual cells or nuclei samples can be labeled with a Cell Multiplexing Oligo (CMO) and then pooled together prior to loading onto a 10x
Genomics chip. The Feature Barcode molecules can be directly captured by the oligos present on the Gel Beads inside a GEM during GEM-RT and subsequently
amplified (see Stepwise Objectives for assay scheme specifics). The amplified DNA generated from the Feature Barcode molecules can be used for Cell Multiplexing
Library Construction. Upon sequencing and processing the data through Cell Ranger, pooled samples can be bioinformatically demultiplexed and analyzed as individual samples, with identified cell multiplets excluded.

Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for Cell Multiplexing.

The labeling cells/nuclei steps are performed in the user's lab using the kit 

3' CellPlex Kit Set A, 48 rxns PN-1000261 

Then bring the labeled cells or nuclei ready to the core lab for cell counting and further processing.

Highlights
1. Sensitive and Unbiased Characterization of
Transcriptional Signatures
Highly sensitive and reproducible gene detection in single
cells
2. Comprehensive Single-Cell RNA Sequencing Workflow
Fully supported workflow developed in collaboration by
technology innovators
3. Powerful Next-Generation Sequencing Integrated with
Simple Data Analysis
Proven Illumina sequencing combined with streamlined, userfriendly
analysis software

This protocol is optimized for cDNA synthesis from 1– 1,000 intact cells or ultra-low input amounts of total RNA. Due to the sensitivity of the protocol, the input material (total RNA or cells) needs to be collected and purified under clean-room conditions to avoid contamination. The whole process of SMART cDNA Synthesis should be carried out in a PCR Clean Work Station under clean-room conditions.

The assay is for preparation of target- enriched, strand- specific RNA sequencing libraries from either high- quality RNA samples or formalin- fixed paraffin- embedded (FFPE) total RNA samples, without initial mRNA purification. This protocol is specifically optimized to enrich targeted regions of the transcriptome from repetitive sequences and sequences unrelated to the research focus prior to Illumina paired- end multiplexed sequencing.

Input total RNA: 100 - 200ng.

The Archer FusionPlex ALK, RET, ROS1 v2 kit is a targeted sequencing assay to simultaneously detect and identify fusions and mutations of human ALK, RET and ROS1 genes. The kit is powered by Archer’s proprietary Anchored Multiplex PCR (AMP™) target enrichment chemistry to detect fusions of all genes in a single sequencing assay, even without prior knowledge of fusion partners or breakpoints.The kit also detects select insertions and point mutations in ALK and RET, including those reported in cell-based assays to convey crizotinib resistance

The Archer FusionPlex Solid Tumor Kit is a robust targeted sequencing assay to simultaneously detect and identify Fusions and other Mutations associated with over 50 genes linked to various carcinomas. Also included in the kit are primers that target known sarcoma- and hematological malignancy-associated Fusions as well as prominent BRAF and PDGFA Mutations.

The power of the FusionPlex Solid Tumor Kit lies in Archer’s proprietary Anchored Multiplex PCR™-based enrichment. This chemistry enables detection of all Fusions associated with the genes in this kit in a single sequencing assay, even without prior knowledge of Fusion partners or breakpoints.

It works with all clinical sample types, including FFPE.

Immunoverse BCR kits amplify B cell receptor heavy (IGH) and light (kappa/lambda) chains for sequencing and analysis. Primers are designed to identify isotypes and subclasses in the Fc region and somatic hypermutations within the variable regions. Sample inputs range from 25ng to 6μg.

The Archer Immunoverse TCR alpha/delta panel is a target-enrichment assay that can be used to create libraries for next generation sequencing of TCR alpha/delta chains. Libraries are created by using the Immunoverse alpha/delta assay in conjunction with the Archer Immunoverse TCR reagents and Archer MBC Adapters.

- Input nucleic acid (Total RNA) in EDTA-free buffer or Ultra-Pure Water is the optimal starting template for Archer AMP Library Preparation. Do not use EDTA-containing buffers exceeding 1mM.

- Use the maximum allowable input mass (ng) whenever possible.

- The minimum recommended input mass for Archer Immunoverse TCR assays is dependent on experiment type, tissue type and tissue quality: o 20ng of RNA for screening of high abundance clones from high-quality PBMC samples o 400ng of RNA for deep sequencing applications from high-quality, highcomplexity PBMC samples o 400ng of RNA from fresh-frozen tissue samples o >400ng for FFPE input for TIL analysis

- The rarefaction analysis below derived from varying amounts of high-quality input from a DNAse-treated PBL sample illustrates the relationship between input amounts, sequencing depths and clonotype count.

-  If using FFPE sample types, we recommend extracting TNA using Promega ReliaPrep™, Agencourt® FormaPure® or Promega Maxwell® RSC RNA FFPE Kit with the modifications to the published manufacturer protocol.

- It is recommended to use the Illumina® MiSeq® 600-cycle kit for the Immunoverse TCR alpha/delta kit.

The Archer Immunoverse TCR beta/gamma panel is a target-enrichment assay that can be used to create libraries for next generation sequencing of TCR beta/gamma chains. Libraries are created by using the Immunoverse beta/gamma assay in conjunction with the Archer Immunoverse TCR reagents and Archer MBC Adapters.

- Input nucleic acid (Total RNA) in EDTA-free buffer or Ultra-Pure Water is the optimal starting template for Archer AMP Library Preparation. Do not use EDTA-containing buffers exceeding 1mM.

- Use the maximum allowable input mass (ng) whenever possible.

- The minimum recommended input mass for Archer Immunoverse TCR assays is dependent on experiment type, tissue type and tissue quality: o 20ng of RNA for screening of high abundance clones from high-quality PBMC samples o 400ng of RNA for deep sequencing applications from high-quality, highcomplexity PBMC samples o 400ng of RNA from fresh-frozen tissue samples o >400ng for FFPE input for TIL analysis

- The rarefaction analysis below derived from varying amounts of high-quality input from a DNAse-treated PBL sample illustrates the relationship between input amounts, sequencing depths and clonotype count.

-  If using FFPE sample types, we recommend extracting TNA using Promega ReliaPrep™, Agencourt® FormaPure® or Promega Maxwell® RSC RNA FFPE Kit with the modifications to the published manufacturer protocol.

- It is recommended to use the Illumina® MiSeq® 600-cycle kit for the Immunoverse TCR alpha/delta kit.

Partial library prep. - if you submit tagmented DNA.

The Illumina DNA Prep protocol uses tagmentation, and enzymatic reaction, to fragment DNA and add adapter sequences and is compatible with DNA inputs of 1–500 ng or higher. For human DNA samples and other large complex genomes, the recommended minimum DNA input is 100–500 ng. For small genomes (eg microbial), the DNA input amount can be reduced to as low as 1 ng (modifying the PCR cycling
conditions accordingly). Assess DNA purity to make sure that the initial DNA sample does not contain > 1 mM EDTA and is free of organic contaminants, such as phenol and ethanol. These substances can interfere with the tagmentation reaction and result in assay failure.

llumina Total RNA Prep with Ribo-Zero Plus is based on TruSeq ligation technology. This ligation method results in high coverage uniformity, precise strand information, and robust and reliable library preparation even from degraded samples. 

The kit supports a broad range of RNA inputs, from 1 ng to 1000 ng. This input range is applicable to high-quality RNA. We recommend 10 ng input for best results and for FFPE or degraded samples.

It’s compatible with variable sample types, including formalin-fixed paraffin-embedded (FFPE) and other low-quality samples. The included Ribo-Zero Plus removes abundant RNA from multiple species, including human, mouse, rat, bacteria, and epidemiology samples.

The QuantSeq FWD Kit is an all-in-one library preparation protocol designed to generate Illumina compatible libraries of sequences close to the 3’ end of polyadenylated RNA.

The kit is ideal for gene expression counting as NGS reads are generated towards the poly(A) tail. To get the exact 3’ transcript end longer reads are necessary.

QuantSeq FWD Kits are delivered with several indexing options:

• Unique Dual Indexing with the Lexogen UDI 12 nt Sets (Cat. 113.96, 129-131.96, 114.96, 115.384) with up to 384 Unique Dual Indices.
• Single Indexing with the Lexogen i7 6 nt Index Sets (Cat. No. 015.24/96/2×96)
• Combinatorial Dual Indexing with the Lexogen i7 6 nt Index Set and i5 6 nt Dual Indexing Add-on Kit (015.384)

This is a combined protocol for using NEBNext Ultra II Directional RNA Library Prep Kit and Twist Exome Kit #2.

Input total RNA: <100ng

PCR amplicons as the input. The input amount: 10ng - 1ug based on which sample preparation protocol is used.

A poly-A based protocol and it contains the strand information. , it requires 25 - 100 ng of starting total RNA in 10-30 µl volume with an RIN value great than 7.8.

Procedure:

This sample preparation protocol that includes rRNA-depletion offers strand information on RNA transcript; Library capture of both coding RNA, as well as multiple forms of non-coding RNA; Degraded RNA can be used with minor adjustments to fragmentation procedures

Starts with total RNA.

Output: 1 M reads / run.

You can perform a single read 50 cycles run.

upload sequencing data to GEO

• This service is only available for internal WCMC researchers.
• We accept the samples for QC including total RNA, cRNA, microRNA, genomic DNA, single stranded cDNA, double stranded cDNA and PCR product.
• Samples should be diluted with molecular biology grade water (RNase-free and Nuclease-free).
• Samples are tested for quality assurance on the Agilent 2100 Bioanalyzer and quantitated on the Nanodrop Spectrophotometer or Invitrogen Qubit Fluorometer as neccessary.
• Users must submit at least 3 µL of sample in a 1.5 mL microfuge tube.
• It is the user's responsibility to dilute the samples to the proper concentration range.

• RNA Quality Control.
• Sample submission: 3 ul (>50 ng/ul)

• RNA Quality Control.
• Sample submission: 3 µl (1-5 ng /µl).

Genomic DNA ScreenTape:

For the analysis of genomic DNA from 200 to > 60,000 bp.

For the analysis of total RNA from 25 to 500ng/µL.

Sizing range: 35 - 1000 bp.

Quantitative Range: 0.1 - 50 ng/µL

High Sensitivity D1000 Reagents:

For the analysis of DNA from 35 to 1000 bp. 

High Sensitivity RNA ScreenTape:

For the analysis of total RNA from 500 to 10,000 pg/µL. 

30Gb / 100M reads / run.

40Gb / 400M reads / run.

80Gb / 400M reads / run.

120Gb / 400M reads / run.

100Gb / 1Billion reads

200Gb / 1 Billion reads

300 Gb / 1 Billion reads

50 GB / 1 Billion reads.

Set up indexing PCR, pool, purification and QC of PCR products before sequencing. Charge $5/well (AOI).