03 Sample Preparation
Quality and quantity of DNA and RNA is critical for high quality sequencing output. Please make sure your DNA is not degraded and is free of RNA contamination. RNA samples should always be assessed on the bioanalyzer for the absence of gDNA contamination (can be removed with DNaseI treatment followed by a column clean-up; e.g. Zymo “RNA Clean and Concentrator”) and degradation. Preferentially determine the concentrations of your DNA and RNA samples using fluorometry (e.g. with a Qubit or plate reader). The sample purity should be assessed by spectrophotometry (e.g. Nanodrop). Please see this page for a comprehensive table of sample requirements for sample QC, library preps, or your self-made libraries. Please see the Library Prep Page for details on the library prep processes. For submission information, including submission forms and shipping details, please visit the Sample Submission & Scheduling page. If you are submitting DNA for PacBio libraries, please follow the PacBio Guidelines for Shipping and Handling.
The Real-time PCR core can carry out DNA as well as RNA extractions for you.
Bead based protocols (e.g. Ampure XP, RNAClean XP) and spin column based protocols (Qiagen, Zymo, NorgenBiotek, …) tend to be the most efficient ways to remove chemical contaminants. For Illumina sequencing we suggest spin column based solutions as the most reliable option. Genomic DNA cleaned up with a spin column further has the advantage that it will always dissolve well.
Multiple protocols are available to remove DNA or RNA contaminations. Please find our suggestions for affordable solutions for Illumina sequencing below.
RNA samples need to be DNA-free (the RNA isolation protocol should always include a DNAse digestion step; in problematic cases you could use RNA-clean & concentrator kits with DNAse). On an agarose gel, DNA contamination will be visible as a smear of band of fragments considerably larger than the RNA (>10 kb). On the Bioanalyzer RNA-chips DNA contamination will be visible in the size range from 4kb to 10 kb.
In case you are using a Trizol protocol for the RNA extractions, we would highly recommend to clean up the samples afterwards with a spin column kit (e.g. RNA-clean & concentrator kits) to make sure to remove any phenol traces.
Please note that the additional column cleanup is mandatory for RNA samples isolated from blood PAXgene or Tempus tubes (for blood sample preservation) or with the accompanying PAXgene and Tempus RNA isolation kits.
DNA samples need to be RNA-free (the DNA isolation protocol should always include a RNAse digestion step; in problematic cases we recommend to use RNAse I (e.g. adding 1 ul RNAse I to your sample and incubate at 30 degrees C for 20 minutes). RNAse I does not require a special buffer ( it works in TE buffer) and can be completely inactivated by heating at 70°C for 15 minutes. Thus, a removal of the enzyme and of a buffer can be avoided in many cases. (For some applications small amounts of RNAse I can remain in the sample without inactivation ore removal — please inquire). For the removal of RNAse I Ampure beads (or similar) or DNA-clean & concentrator kits will work fine. DNA samples can be QC-ed easily by agarose gel electrophoresis and ethidiumbromide staining. The stain will make both DNA and RNA visible. RNA will run as an halo-like smear in the range of 50- to 200 bp.
- The fragment length should be between 100 and 300 bp (up to 400 for the majority of molecules is acceptable).
- Please make sure to run the input controls on the bioanalyzer or on an agarose gel beforehand and email us an image of these.
- Sequence one “input control” per cell line/ sample type.
- It is highly recommended to verify the enrichment of your regions of interest (e.g. promoter regions) vs. the control samples by qPCR, before submitting the samples for sequencing.
The required read-number per sample will vary from target to target. For the study of point source transcription factors the ENCODE project recommends to analyze at last 20 million (uniquely mapping) reads ( http://genome.cshlp.org/content/22/9/1813.long#boxed-text-2 ). Depending on the quality of your preps perhaps 75% of the reads can be expected to be uniquely mapping. ENCODE tends to err on the high side with their recommendations. Thus about 20 million reads per sample should be acceptable but this is likely the minimum number.