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Next Generation Sequencing

Transcriptomics

mRNA Sequencing (Gene Expression, isoform analysis)

If providing tissue or cells, we require enough material to obtain at least 0.2 - 0.5ug (200-500ng) of total RNA per sample.  For purified RNA, we require submitting at least 0.250ug (250ng) of purified total RNA in at least 5ul of ultra pure, nuclease-free water. DNase treatment of RNA is strongly recommended and can be done prior to submission using gDNA eliminator columns (Qiagen), Turbo DNase (Life Technologies) or on-column DNASE treatment.  The GRC is able to perform DNASE treatments for additional fees. Sample quantity and quality will be verified with spectrophotometry (NanoDrop 1000), fluorometry (Qubit), and the Agilent Bioanalyzer 2100 profile.  RIN (RNA Integrity Number) values of >7.0 and OD260/280 = 2.0 - 2.2 are recommended for RNA-seq library preparation.   Please fill out the submission form in its entirety and place samples at -80C located in the GRC Drop-off room on the Medical Campus.  It is critical for purified RNA to be transported on ice or dry ice (if frozen) to prevent degradation.

Low Input RNA Sequencing (Gene Expression, isoform analysis)

If providing tissue or cells, we require enough material to obtain at least 5ng of total RNA per sample.  For purified RNA, we require submitting at least 5ng of purified total RNA in at least 5ul of ultra pure, nuclease-free water. DNase treatment of RNA is strongly recommended and can be done prior to submission using gDNA eliminator columns (Qiagen), Turbo DNase (Life Technologies) or on-column DNASE treatment.  The GRC is able to perform DNASE treatments for additional fees. Sample quantity and quality will be verified with spectrophotometry (NanoDrop 1000) and the Agilent Bioanalyzer 2100 profile.  RIN (RNA Integrity Number) values of >7.0 and OD260/280 = 2.0 - 2.2 are required for low input RNA-seq library preparation.   Please fill out the submission form in its entirety and place samples at -80C located in the GRC Drop-off room on the Medical Campus.  It is critical for purified RNA to be transported on ice or dry ice (if frozen) to prevent degradation.

Total RNA Sequencing (Gene Expression, isoform analysis, non-coding RNA, De novo assembly)

If providing tissue or cells, we require enough material to obtain at least 0.2 - 0.5ug (200-500ng) of total RNA per sample.  For purified RNA, we require submitting at least 0.250ug (250ng) of purified total RNA in at least 5ul of ultra pure, nuclease-free water. DNase treatment of RNA is required and can be done prior to submission using gDNA eliminator columns (Qiagen), Turbo DNase (Life Technologies) or on-column DNASE treatment.  The GRC is able to perform DNASE treatments for additional fees. Sample quantity and quality will be verified with spectrophotometry (NanoDrop 1000), fluorometry (Qubit), and the Agilent Bioanalyzer 2100 profile.  RIN (RNA Integrity Number) values of >7.0 and OD260/280 = 2.0 - 2.2 are recommended for RNA-seq library preparation.   Please fill out the submission form in its entirety and place samples at -80C located in the GRC Drop-off room on the Medical Campus.  It is critical for purified RNA to be transported on ice or dry ice (if frozen) to prevent degradation.

Small RNA Sequencing (micro RNA)

If providing tissue or cells, we require enough material to obtain at least 1-2ug of total RNA. For purified RNA, we recommend submitting at least 1-2ug of purified total RNA (including small RNA) or enriched small RNA (<200nt) from 1-2ug total RNA (~50ng) in at least 5ul of ultra pure, nuclease-free water.  DNase treatment of RNA is necessary and can be done prior to submission using Turbo DNase or the GRC will perform this for a fee. Sample quantity and quality will be verified with spectrophotometry (NanoDrop 1000), fluorometry (Qubit), and the Agilent Bioanalyzer 2100 profile.   Please fill out the submission form in its entirety and place your samples in -80C located in the GRC Drop-off room on the Medical Campus.  It is critical for purified RNA to be transported on ice or dry ice (if frozen) to prevent degradation.

Single-Cell RNA Sequencing (scRNAseq)

The GRC currently is capable of providing scRNAseq library and analysis support using Fluidigm C1, 10X Genomics, and FACS-based methods.  For more information, please contact us.

Genomics

Whole Genome Sequencing (WGS)

If providing tissue, blood or cells, we require enough material to obtain at least 2ug of total gDNA.  For purified gDNA, we recommend submitting at least 2ug of purified total gDNA in at least 10ul of ultra pure, nuclease-free water.  Sample quantity and quality will be verified with spectrophotometry (NanoDrop 1000), fluorometry (Qubit), and Agilent TapeStation 2200. We recommend DNA purity with an OD260/280=1.8-2.0 with high molecular weight (no degradation) or RNA contamination. Please fill out the submission form in its entirety and place your samples in -80C located in the GRC Drop-off room on the Medical Campus.

Exome Sequencing

If providing tissue, blood or cells, we require enough material to obtain at least 1ug of total gDNA.  For purified gDNA, we recommend submitting at least 1ug of purified total gDNA in at least 10ul of ultra pure, nuclease-free water.  Sample quantity and quality will be verified with spectrophotometry (NanoDrop 1000), fluorometry (Qubit), and Agilent TapeStation 2200. We recommend DNA purity with an OD260/280=1.8-2.0 with high molecular weight (no degradation) or RNA contamination. Please fill out the submission form in its entirety and place your samples in -80C located in the GRC Drop-off room on the Medical Campus.

Gene Regulation

ChIP Sequencing

Please submit at least 20-50ng of ChIP or capture kit enriched DNA in ultra pure, nuclease-free water.  We strongly recommend ChIP DNA be fragmented to 200-600bp in size for optimal library performance and reduced background noise levels.  Sample quantity and quality will be verified with spectrophotometry (NanoDrop 1000), fluorometry (Qubit), and the Agilent Bioanalyzer 2100 profile. We recommend DNA purity with an OD260/280=1.8-2.0 without RNA contamination. Please fill out the submission form in its entirety and place your samples in -80C located in the GRC Drop-off room on the Medical Campus.

Epigenomics

Whole Genome Bisulphite Sequencing (WGBS)

If providing tissue, blood or cells, we require enough material to obtain at least 1ug of total gDNA.  For purified gDNA, we recommend submitting at least 1ug of purified total gDNA in at least 10ul of ultra pure, nuclease-free water.  Sample quantity and quality will be verified with spectrophotometry (NanoDrop 1000), fluorometry (Qubit), and Agilent TapeStation 2200. We recommend DNA purity with an OD260/280=1.8-2.0 with high molecular weight (no degradation) or RNA contamination. Please fill out the submission form in its entirety and place your samples in -80C located in the GRC Drop-off room on the Medical Campus.

Reduced Representation Bisulphite Sequencing (RRBS)

If providing tissue, blood or cells, we require enough material to obtain at least 1ug of total gDNA.  For purified gDNA, we recommend submitting at least 1ug of purified total gDNA in at least 10ul of ultra pure, nuclease-free water.  Sample quantity and quality will be verified with spectrophotometry (NanoDrop 1000), fluorometry (Qubit), and Agilent TapeStation 2200. We recommend DNA purity with an OD260/280=1.8-2.0 with high molecular weight (no degradation) or RNA contamination. Please fill out the submission form in its entirety and place your samples in -80C located in the GRC Drop-off room on the Medical Campus.

MeDIP Sequencing

Methods currently under evaluation ...

ATAC-seq

Through collaboration with Laurie Steiner, MD, and Michael Bulger, PhD the GRC is able to provide ATAC-seq library construction, sequencing, and analysis.  For more information, please contact us.

Targeted Resequencing

If providing tissue, blood or cells, we require enough material to obtain at least 500ng of total gDNA.  For purified gDNA, we recommend submitting at least 500ng of purified total gDNA in at least 10ul of ultra pure, nuclease-free water.  Sample quantity and quality will be verified with spectrophotometry (NanoDrop 1000), fluorometry (Qubit), and Agilent TapeStation 2200. We recommend DNA purity with an OD260/280=1.8-2.0 with high molecular weight (no degradation) or RNA contamination. Please fill out the submission form in its entirety and place your samples in -80C located in the GRC Drop-off room on the Medical Campus.

Metagenomics

16S rRNA Amplicon Sequencing

If providing tissue, blood or cells, we require enough material to obtain at least 500ng of total gDNA.  For purified gDNA, we recommend submitting at least 500ug of purified total gDNA in at least 10ul of ultra pure, nuclease-free water.  Sample quantity and quality will be verified with spectrophotometry (NanoDrop 1000), fluorometry (Qubit), and Agilent TapeStation 2200. We recommend DNA purity with an OD260/280=1.8-2.0 with high molecular weight (no degradation) or RNA contamination. Please fill out the submission form in its entirety and place your samples in -80C located in the GRC Drop-off room on the Medical Campus.

Dual RNA-seq

If providing tissue or cells, we require enough material to obtain at least 1ug of total RNA per sample.  For purified RNA, we require submitting at least 1ug of purified total RNA in at least 10ul of ultra pure, nuclease-free water. DNase treatment of RNA is required and can be done prior to submission using gDNA eliminator columns (Qiagen), Turbo DNase (Life Technologies) or on-column DNASE treatment.  The GRC is able to perform DNASE treatments for additional fees. Sample quantity and quality will be verified with spectrophotometry (NanoDrop 1000), fluorometry (Qubit), and the Agilent Bioanalyzer 2100 profile.  RIN (RNA Integrity Number) values of >7.0 and OD260/280 = 2.0 - 2.2 are recommended for RNA-seq library preparation.   Please fill out the submission form in its entirety and place samples at -80C located in the GRC Drop-off room on the Medical Campus.  It is critical for purified RNA to be transported on ice or dry ice (if frozen) to prevent degradation.

Sequence-Ready Libraries (Non-GRC prepared)

All sequence-ready libraries should be submitted in clearly labeled nuclease-free 1.5ml low-bind microfuge tubes along with the completed "User-Prepared Library" submission form.  The GRC offers 3 library QC options:

A) Library pools submitted as 2nM stocks to GRC in EB buffer.  Libraries should be pooled based on lane usage prior to submission at 2nM.  The GRC will load these library pools directly on the sequencer assuming 2nM concentration.

B) Sample libraries will be processed through our standard library QC (Qubit, Bioanalyzer, KAPA qPCR), normalized to 2nM,  and pooled per GRC standard protocols for sequencing.  **Note:  This QC does not guarantee successful sequencing or optimal cluster generation as it does not account for contribution of incomplete adaptor ligation or excess adaptor present, both of which negatively impact flow cell hybridization and cluster generation.**

C) Sample libraries will be processed through our standard library QC (Qubit, Bioanalyzer, KAPA qPCR), normalized to 2nM, pooled per GRC standard protocols for sequencing, and a test run will be performed using the MiSeq.  This directly assesses library sequencing performance which the data will be used to correct the pooling strategy and adjust target concentration for the HiSeq2500 sequencing run.  **Note: Due to indexing, each lane submitted will be assessed by an independent MiSeq run.**