Bulk Short-Read RNA-seq Library Preparation Services

We provide RNA library preparation services for a variety of RNA sample types (total RNA, reduced rRNA, small RNA, Ip RNA, and mRNA). We use a variety of commercial kits to prepare RNA libraries that have been tested in-house on multiple sample types.

For new clients we recommend a meeting is  scheduled with us to discuss your project before submission.

RNA quality recommendations:

1. RNA samples should be DNase treated.
2. A₂₆₀/A₂₈₀ ratio range 1.8 – 2.1; A₂₆₀/A₂₃₀ ratio should be similar indicating pure nucleic acids.  Inaccurate ratios may also be encountered at very low concentrations (< 10 ng/µl) of nucleic acids.
3. We strongly recommend the absorbance spectrum of each RNA sample is visually checked to ensure the peak is directly over 260nm.  Residual chemical contamination from nucleic acid extraction procedures may result in an overestimation of the nucleic acid concentration and/or negatively influence downstream analysis.
4. RNA concentration should be 30-300ng/uL; if >300ng/uL please dilute.
5. RNA with concentration < 10ng/uL, sample should be quantified on a fluorometer for a more accurate concentration.
6. Agilent RIN^e > 7.0.  The RNA Integrity Number (RIN^e) algorithm is based on a mathematical model that calculates an object quantitative measurement of RNA degradation. RIN^e represents the relative ratio of the signal in the fast zone to the 18S peak signal. Highest quality RNA is assigned a RIN^e value of 10. Calculated automatically by the 4200 TapeStation software, RIN^e provides an objective and reliable numerical assessment of the integrity of eukaryotic/prokaryotic total RNA degradation.

RNA INPUT range per library prep type:

NEBNext Ultra II Directional RNA  25-1,000ng total RNA* (includes both mRNA & rRNA reduction)

TruSeq Stranded RNA 100-1,000ng total RNA* (includes both mRNA & rRNA reduction)

Takara SMARTer Stranded Total RNA Seq v2 Pico Input Mammalian  1-10ng total RNA *

QIASeq miRNA  cells/tissue 1-500ng total RNA (100ng optimal); serum/plasma 5uL from 200uL  serum/plasama extraction (miRNeasy Serum/Plasma Kit or miRNeasy Serum/Plasma Advanced Kit); exosomes 5uL from 1mL serrum/plasma extraction (exoRNeasy Kit )

*when possible we prefer to target a higher INPUT to minimize the number of PCR cycles needed to ensure sufficient yeilds for sequencing

SAMPLE SUBMISSION

• Sample IDs must be alphanumeric with 3-20 characters. Dashes (-) are allowed.  No spaces or duplication of names.
• RNA projects with ≥8 samples, samples must be submitted in a sealed -80C thermostable clear full or semi skirted v-bottom 96-well plate. Each plate must be filled by column (A1-H1, etc.).  Plates filled by row (A1-A12) will be returned to client at $150 charge.
• Plates must be labeled with

1. Submission Number generated with the service request (M00xxxx)
2. PI name
3. Date

A $75 service charge will be incurred for incomplete plate labeling.

• RNA projects with <8 samples, samples may be submitted in a 1.5mL tube with the sample ID on the lid and PI name and date on the side. Failure to follow this will result in a $75 service charge.  The sample ID on the lid must match the Sample Submission sheet.
• We require a minimum of 15uL per sample as 3-4uL is used for RNA QC. If the RNA is suspended in something other than nuclease-free water, please include an aliquot when submitting your samples.
• QC of RNA will occur within 1-3 business days.  QC will be by NanoDrop and Agilent to confirm conc./purity and integrity, respectively.  Results will be returned to client with comments/suggestions.
• Remaining template may be returned to client upon request.
• RNA will be discarded six months post project completion.

DATA ANALYSIS

The UWBC Bioinformatics Resource Center offers fee for service data analysis options.

ACKNOWLEDGING UNIVERSITY OF WISCONSIN-MADISON BIOTECHNOLOGY CENTER

A visible measure of a core’s impact is made through proper acknowledgement of usage in abstracts/talks/publications. To acknowledge our core, please use the following statement:

“The author(s) utilized the University of Wisconsin – Madison Biotechnology Center’s Gene Expression Center (Research Resource Identifier – RRID:SCR_017757) for RNA library preparation.