Transcriptomics

RNA-Seq (RNA sequencing), also called whole transcriptome shotgun sequencing (WTSS), uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment in time.

RNA-Seq is used to analyze the continuously changing cellular transcriptome. Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression over time, or
differences in gene expression in
different groups or treatments. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, and small RNA, such as miRNA.

The RNA-Seq workflow should start with experimental design (GGBC consultation). After designing the experiment, a suitable method is chosen to isolate and purify RNA. GGBC accepts purified total RNA as the input to a range of NGS library preparations. For each library preparation, the workflow starts with the quality assessment of the input RNA. The RNA quality is critical for successful RNA-Seq experiment.  is critical for maximizing the quality of the returned data.

Consultation and Assistance

Contact for Genomics and Bioinformatics Consultation

Please use this link to send an email to Dr. Magdy Alabady for consultation on new or existing Genomics and Bioinformatics projects. Also, you can contact Dr. Alabady for assistance with grant proposals and for obtaining a letter of support from GGBC.

For technical questions about existing or new Illumina projects, please contact the following Lab Technician:

More contacts

Please visit our “All Inquiries” page for detailed information about who you should contact at GGBC to receive a quick and accurate response.

Sample Preparation

Libraries prepared with the following sample preparation kits at the GGBC can be used on any of Illumina’s next generation sequencing instruments including MiSeq, NextSeq 500 and HiSeq.

Crucial recommendations for all samples

  • Use fluorometric based methods for quantification (Qubit or PicoGreen) of the template DNA instead of UV spec based methods (e.g., NanoDrop) to obtain accurate DNA measurement.
  • DNA should have absorbance ratio values of 1.8–2.0.
  • RNA samples can be suspended in RNase-free water or 1X TE buffer prepared with RNase-free water.
  • RNA integrity should be assessed using the Agilent Bioanalyzer (or any similar system).
  • Please add any QC information (Qubit data, Agilent run, fragment analyzer run, gel picture,…) available to the online order form.
  • If you are are submitting 24 or more samples, please submit samples in a 96-well plate and include an Excel file with the sample layout in your order.
  • PLEASE USE V BOTTOM PLATES AND NOT ROUND/FLAT BOTTOM PLATES.

NGS stranded or unstranded RNA library preparation (Illumina compatible)  

In the stranded libraries the vast majority of the sequencing reads are from the first strand.  Selecting certain library types depends on the application. Generally speaking, all expression-by-sequencing analyses should use stranded libraries. Non-stranded RNA libraries can also be prepared upon request.  Feel free to contact us at ggbc@uga.edu to discuss your project.

Currently at GGBC, we use the Kapa Biosystems RNA library preparation chemistry for constructing of stranded libraries. We prepare both single- and dual-indexed libraries, depending on the target level of multiplexing. The quality of starting RNA is crucial for making good libraries and most importantly for robust data analysis. Starting material can be:

  •  100 ng – 4 μg of total RNA in < 50 µl
  •  10 – 400 ng of rRNA depleted or poly(A) – enriched RNA

RNA samples can be suspended in RNase-free water or 1X TE buffer prepared with RNase-free water. RNA integrity should be assessed using the Agilent Bioanalyzer (or any similar system). For sample’s concentration, we prefer using fluorometric methods, such as Qubit or Ribogreen.

NGS ribo-depleted stranded or unstranded RNA library preparation (bacteria)

Treatment of the total RNA from bacteria with the Ribo-Zero protocol removes cytoplasmic (nuclear-encoded) rRNA (5S, 16S, and 23S) prior to strand-specific or unstranded library preparation and sequencing. Please provide 1-5 µg of total RNA in RNase-free water.

TruSeq small RNA library preparation 

The TruSeq Small RNA sample preparation kit primarily targets microRNAs and other small RNAs, that have a 5’-phosphate and a 3’-hydroxyl group, to generate cDNA from total RNA or purified small RNA. Up to 48 samples can be multiplexed in one sequencing lane. Please provide 1 to 20 μg of high-quality total RNA prepared using a method that retains the small RNA fraction (Trizol etc.) at a concentration of at least 250 ng/µl in high quality water or 10 mM Tris buffer. Alternatively, submit the entire fraction of small RNA purified from 5-20 μg of total RNA in molecular grade water or 10 mM Tris buffer. Starting with enriched small RNA sample decreases the background signals, as it filters out most of the RNA degradation products.This kit supports only single-indexed libraries at the moment (up two 48 barcodes are available). RNA samples can be suspended in RNase-free water or 1X TE buffer prepared with RNase-free water. RNA integrity should be assessed using the Agilent Bioanalyzer (or any similar system). For sample’s concentration, we prefer using fluorometric methods, such as Qubit or Ribogreen.

Ready-to-run libraries

Please provide ≥20 µl of your ready to run library at a 10 nM concentration in 10 mM Tris pH 8. If you would like to use a custom primer for your MiSeq run, please submit ≥80 µl at a concentration of 10 mM. Please add any QC information (Qubit data, Agilent run, fragment analyzer run, gel picture,…) available to the online order form.

SMARTer Pico cDNA Synthesis

Synthesis of cDNA from 1-2 ng of total RNA. Minimum total RNA concentration for this cDNA synthesis kit is 20pg/uL in a 50uL reverse transcription reaction.

Fragment Analyzer QC: Electropherogram Reading and interpretation 

Fragment Analyzer QC: Electropherogram Reading and interpretation

 

RNA is a sensitive material. RNA should be kept on dry or wet ice during transportation to the GGBC. Wet ice is recommended only for samples that are being transported locally within Athens. If you are traveling from farther away to submit your RNA samples, keep them on dry ice during transit. The GGBC will not accept RNA samples that are not stored properly during transit.

RNA Quality Assessment

Below are some example Agilent Bioanalyzer data sets from high quality RNA extractions submitted to GGBC.

Plant tissue from roots and leaves

Insect RNA with co-migrating 18S and 28S left and right arm

Eukaryotic RNA

Prokaryotic RNA (Salmonella)

Small RNA

RNA-seq Libraries

The GGBC currently prepares mRNA-Seq libraries from poly-adenylated total RNA for the Illumina platform using a strand orientation preserving mRNA-Seq method (stranded). Polyadenylated mRNA is first captured with oligo (dT)n modified paramagnetic beads, washed to remove unbound RNA and eluted into a fragmentation solution. After fragmentation with heat in the presence of metal ions, randomly primed first strand synthesis is initiated. For second strand marking and strand orientation preservation, dUTP is incorporated during the second strand synthesis. End repair, A-tailing and Illumina adapter ligation followed by amplification with a dUTP intolerant polymerase completes the library synthesis.

Anthor RNAseq approach is the 3’ mRNA-seq method. The first strand synthesis is initiated from the mRNA polyadenylated tail providing sequences adjacent to the 3’UTR. The method imposes some restrictions on the type of sequencing you can do on the Illumina platforms and biases the sequenced molecules toward the 3’ end of the RNA but it simplifies the analysis and is more cost effective.

For non-polyadenylated RNA and for samples where the analysis of non-polyadenylated RNAs is desired, the GGBC uses the Illumina Ribozero rRNA depletion reagents to remove the structural RNA. The standard RNA-Seq protocol is then initiated at the fragmentation and randomly primed first strand synthesis step.

The small RNA-seq library are prepared using a gel-free method starting with total RNA as input. Truncated Illumina specific adapters are directly ligated to the 3’ OH group and the 5’ phosphate group of the small RNAs using modified RNA ligases, followed by reverse transcription and PCR. The gel-free method is more robust and cost effective than other gel-based methods.