RNA-Seq

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Overview

RNA-Seq is an application used to sequence the transcriptome of mRNA or small RNAs using Next-Generation Sequencing, to identify transcripts expressed at a given time.

RNA extraction protocol
  1. Excellent RNA quality is an essential parameter for the success of RNA-Seq. Total RNA can be extracted using common lab reagents such as Clontech Nucleospin RNA kits, Qiagen RNAeasy kits, Zymo kits (Direct-zol or Quick-RNA miniprep kits) or Trizol from Invitrogen .For suggested RNA sample prep, see full protocol.
  2. Please include DNase treatment your RNA to eliminate background genomic DNA contamination. An example kit (one of many) is Turbo DNase or DNA Removal Kit (ThermoFisher). Clontech and Zymo include DNase in their kits. Please use RNA purification columns (e.g. Zymo) or ethanol precipitation to eliminate residual enzymes and DNase buffer (they were shown to interfere with downstream processes).
  3. Resuspend or elute your RNA in RNase-free water to minimally 15uL (starting volume for Total RNA-Seq library prep is 10uL, Stranded mRNA-Seq library prep is 50uL).
  4. Store your samples under appropriate RNase-free conditions. To avoid freezing and thawing of your RNA, please make a 3uL aliquot in PCR strip tubes for QC after RNA isolation, and freeze down each of your stock RNA in 2 low-binding RNase-free 1.5mL eppendorfs in -80C (one for the RNA-Seq and the other for validation.
  5. IMPORTANT: Label your samples using your initials, sequential numbers and Date.
  6. Email Orlyyaron@post.tau.ac.il your completed Sample Form. Once approved, please coordinate to drop off your samples on dry ice. Run on 1% agarose gel or if there are low RNA concentrations, we can measure RNA integrity number (RIN number) using Agilent Bioanalyzer or Tape Station. If needed, we can measure the sample concentrations using Qubit. Samples with low RIN numbers and/or low concentrations may result in failed RNA-Seq library preps or poor data from RNA-seq. Nanodrop often overestimates RNA concentration.
  7. Once QC of your RNA is done, the Genomic Analysis Lab will proceed with samples that meet the standard (500ng / sample, RIN >7). If your samples do not meet the QC requirements but you’d still like to proceed, you will responsible for the cost for failed preps.
  8. Please save aliquots of your RNA if you need it for other assays. Due to limiting storage space, leftover RNA and libraries will be discarded 3 months after sequencing is finished.
Sample requirements
RNA samples:

 

     Library Preparation Type Input Material Min. Amount Volume Dilution Buffer
  • TruSeq RNA
 Total RNA    1 ug   < 50 ul H2O
  • Small RNA
 Total RNA    1 ug   5 ul H2O

 

Sequencing
At this time, TAU's Genomics Analysis Lab does not perform sequencing in-house. We prepare and submit libraries to outisde laboratories, such as the Technion Genome Center and G-INCPM, where the sequencing is performed.
Costs

Please contact Dr. Orly Yaron in the Functional Genomics Lab at  03 640 5251 or orlyyaron@post.tau.ac.il for pricing information.

Analysis options

The preliminary analysis from raw datafiles is performed on various platforms such as the Partek Genomics Suite. Further analysis of differentially expressed gene-lists function and pathways is performed using various updated web-based tools. The analysis is generally done on a collaborative basis. For more details, please contact Dr. Metsada Pasmanik-Chor: metsada@post.tau.ac.il , 03 640 6992.

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