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 15ul 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.
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 15ul 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.
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 15ul 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.
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 15ul 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.
Single-Cell RNA Sequencing (scRNAseq)
The GRC currently is capable of providing scRNAseq library and analysis support using 10X Genomics and FACS-based methods. If submitting post GEM cDNA, please send in all cDNA for QC and downstream library preparation. If sending user-prepared 10X libraries, please reach out to discuss volume requirements for sequencing. For more information, please contact us. Please visit our single cell webpage for more information on the service.
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 15ul 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.
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 15ul 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.
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.
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.
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.
MeDIP Sequencing
Methods currently under evaluation ...
ATAC-seq
The GRC currently is capable of providing ATAC-seq library and analysis support. 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.
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.
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.
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 form. The GRC offers 3 library QC options:
A) Library pools submitted as 2nM stocks to GRC in ultra pure, nuclease free water. Libraries should be pooled based on flow cell 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.** Please send approximately 30uL at 3nM to allow for QC and proper dilutions for sequencing.
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 NextSeq or NovaSeq sequencing run. **Note: Due to indexing, each lane submitted will be assessed by an independent MiSeq run.**