Samples preparation guidelines
Total RNA for RNA-seq
General indications for RNA submission
In order to obtain a high quality sequencing data, customers must provide a good quality RNA, in detail: · the 260/280 ratio of your RNA sample should be >1.8; · RNA samples should be resuspended in nuclease-free water; · on a gel, high-quality RNA should have two prominent bands (e.g. ribosomal RNA) with the 28S one (at 4.5 kb) should be twice the intensity of 18S (at 1.9 kb); · on an Agilent Bioanalyzer 2100, RNA should have an RNA Integrity Number (RIN) > 8.
RNA samples must be free of contaminating proteins and other cellular material, organic solvents (including phenol and ethanol) and salts. We strongly recommend commercial kits for totalRNA or miRNA extraction (e.g. Spectrum Plant Total RNA Kit, TRI-REAGENT, RNeasy, MirVANA or MirPremier). Customers need to provide the result analysis of Agilent 2100 Bioanalyzer or, at least, gel-electophoresis image that can show the RNA quality. Quantify your RNA samples by spectrophotometer (e.g. Nanodrop) or fluorimeter (e.g. QuBit). Sample acceptance is based on the amount, concentration and Bioanalyzer QC measured by us. Send RNA samples in 1.5-2ml Eppendof tubes. 0.2 and 0.5ml tubes as well as strip tubes WILL BE NOT ACCEPTED. If you have more than 24 samples, please put your samples in a 96-wells plate (MANDATORY). The tubes must have, on the vial top, a clear and permanent sign (or a thin label) with the progressive number of the mailed samples according to the LABEL_ID in the "shipment file". Send RNA samples in dry-ice or lyophilized with RNAstable (Biomatrica).
Requirements protocol-specific
Total RNA for TruSeq Stranded mRNA-seq
The total amount requested is 4 micrograms in at least 100µl (minimum concentration of 40 ng/µl). Use of degraded RNA can result in low yield, over-representation of 3’ends of the RNA molecules or failure of the protocol.
Total RNA for Stranded RNA-seq with one of these modules:
· Ribo-Zero Human/Mouse/Rat · Ribo-Zero Gold Human/Mouse/Rat · Ribo-Zero Plant system · Ribo-Zero Gram-Positive Bacteria
· Ribo-Zero Gram-NegativeBacteria · Ribo-Zero Yeast
The total amount requested is 1 microgram in at least 20 µl (minimum concentration of 50 ng/µl). This protocol works also with degraded RNAs and FFPE RNAs even if the success rate is not guarantee. A DNase I step is mandatory after the RNA isolation. RNA that has DNA contamination will result in an underestimation of the amount of the RNA used and poor data quality. Look at species compatibility here
Total RNA for smallRNA-seq
The total amount requested is 2 micrograms (minimum concentration of 200 ng/µl) of total RNA or 100 ng of previously isolated microRNA (minimum concentration of 10 ng/µl) in 10 µl of nuclease-free water or 10 nM Tris-HCl, pH 8.5.
Total RNA for amplification with Nugen ‘Ovation RNA-Seq v.2’
The total amount requested is 500 pg-100 ng in at least 10 µl (minimum concentration of 100 pg/µl).
DNA for BS-seq and RRBS-seq
Prepare the DNA following your favorite extraction method, even if we strongly recommend to use commercial column-based protocols. Control your DNA on 0.8% agarose gel to check for integrity. For plant DNA it is important to start from young leaves and prepare DNA starting from nuclei in order to avoid an excess of chloroplast DNA contamination.
Submit 100ng of DNA resuspended in WATER at a minimum concentration of 10 ng/µl, majority of the fragments should be >20Kbp (perform a 0.8% agarose gel check your DNA to control for possible degradation).
For every sample received we confirm the amount and concentration by a QBit fluorimeter assay and volume check. Sample acceptance is based on the amount and concentration measured by us, which is often different from the values provided by customers. Mail DNA samples in 1,5-2ml Eppendorf tubes. 0,5ml tubes will be not accepted. If you have >24 samples please put you samples in a 96-wells plate. The tubes must have, on the vial top, a clear and permanent sign (or a thin label) with a progressive number of the mailed samples and the customer’s name (at least the initials). Send DNA samples in a cold pack (e.g. Blue ice).