All submitted material should be suspended in molecular grade water, or 10 mM Tris pH 8.0, never in TE (EDTA is known to inhibit downstream enzymatic reactions). RNA samples should be DNase-treated prior to submission or this service may be requested. We highly recommend kits that have DNase-inhibition reagents that do not contain EDTA, such as Ambion DNA-free DNA removal kit (cat # AM1906). Likewise, DNA samples should always be RNase-treated prior to submission.
We ask for at least 30uL volume per sample regardless of sample type.
All submissions are subject to internal quality controls and they must meet minimum quality requirements prior to library preparation.
Quality controls ensure that all samples perform according to chemistry requirements. To save time and resources, we highly recommend that you run quality controls on your samples prior to sending your samples to our facility.
We recommend quantitating input material using fluorescent methods such as Qubit Quant-it dsDNA HS and RNA assay (Invitrogen) prior to submission. NanoDrop quantitation is not accurate, although absorbance ratios can be useful in evaluating the purity of your starting material (see absorbance ratios technical notes in the ‘Technical Bulletin’ section below).
Upon arrival at our facility, we verify the integrity of all samples by running our own internal controls. Genomic DNA samples are checked on a gel and assessed for intact, high molecular weight material. All other samples are subject to Agilent Bioanalyzer quality assessment, and fluorescent quantitation. Clients are contacted if samples do not meet minimum specifications. For example, RNA samples that have RNA Integrity Number (RIN) of less than 8, are placed on hold, and the customer is contacted for further instructions.
MINIMUM REQUIRED AMOUNTS
We require the following minimum DNA amounts (RNase-treated if applicable, and high quality) for each of the listed library preparation methods:
- Nextera XT kit sample preparation- 2 ng required, recommended minimum 100 ng
- Nextera DNA kit sample preparation- 50 ng required, recommended minimum 100 ng
- Nextera Mate Pair sample preparation- 1-4 ug required, recommended minimum 2-8 ug
- Nextera Rapid Capture Exome sample preparation- 50 ng required, recommended minimum 100 ng
- Illumina TruSeq Exome enrichment protocol- 1 ug required, recommended minimum 2 ug
- Mate-Pair – 20 ug recommended
- ChIP-Seq – 10 ng required, recommended minimum 20 ng
- Nano gDNA sample preparation- 100 ng required, recommended minimum 500 ng
- PCR-free gDNA sample preparation- 1ug required, recommended minimum 2 ug
- Amplicon sample preparation- 250 ng required, recommended minimum 500 ng
We require the following minimum RNA amounts (DNase-treated, and high quality) for each of the listed library preparation methods:
- Standard and stranded mRNA and total RNA sample preparation- 100ng-1ug total RNA required, recommended minimum 4 ug total RNA
- Small RNA sample preparation-1ug total RNA required, recommended minimum 4 ug total RNA
- Illumina TruSeq RNA Access sample preparation (FFPE)- 10ng total RNA required, recommended minimum 20 ng total RNA
We require a minimum of 40 nmoles (in 30 uL at a minimum) of adapter/primer-free library suspended in molecular grade water, or 10 mM Tris pH 8.0, never in TE. We recommend NEBNext Library Quant kit (NEB, cat# E7360) for pooling and for final yield assessments.
Please note that the DSC does not guarantee library performance of customer prepared libraries, nor do we guarantee performance of challenging samples (high GC/AT content), those generally considered a challenge for the current Illumina sequencing technology. Please see our ‘Terms & Conditions’ section for more information.