TITLE: Bulk RNAseq - Mouse Adipose Tissue - University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Bulk RNA sequencing is the method of choice for transcriptomic analysis of pooled cell populations, tissue sections, or biopsies. It measures the average expression level of individual genes across hundreds to millions of input cells and is useful to get a global idea of gene expression differences between samples.

The following protocol was used to isolate, purify and concentrate RNA from murine adipose tissue for Bulk RNAseq analysis. Library preparation and sequencing was completed in collaboration with the University of Minnesota Genomics Center. 

  
  



[STEPS]
SECTION: RNA Isolation (from Thermo Fisher TRIzolReagent User Guide)
1. Lyse and homogenize samples in TRIzol Reagent according to the starting material (adipose tissue). 
Add 1 mL of TRIzol Reagent per 50-100 mg of tissue to the sample and homogenize using a homogenizer. 
SECTION: RNA Sequencing
10. Cluster generation and sequencing:  Pooled libraries are denatured and diluted to the appropriate clustering concentration. Denatured and diluted libraries are loaded onto the NextSeq 2000 cartridge and clustering occurs onboard the instrument.  Once clustering is complete, sequencing immediately commences using Illumina’s 2-color SBS chemistry. Upon completion of read 1, an index read 1 of varying length is performed depending on the library kit used.  If libraries are dual indexed, a second index read is performed. Finally, the library fragments are re-synthesized in reverse orientation and sequenced from the opposite end of the read 1 fragment to produce the paired end read 2.  
SECTION: RNA Isolation (from Thermo Fisher TRIzolReagent User Guide)
2. Centrifuge the lysate for 5 minutes at 12,000 x g at 4-10ºC, then transfer the clear supernatant to a new tube. 

SECTION: RNA Isolation (from Thermo Fisher TRIzolReagent User Guide)
3. Incubate for 5 minutes to allow complete dissociation of the nucleoproteins complex. 
SECTION: RNA Isolation (from Thermo Fisher TRIzolReagent User Guide)
4. Add 0.2mL of chloroform per 1 mL TRIzol Reagent used for lysis, securely cap the tube, then thoroughly mix by shaking. 
SECTION: RNA Isolation (from Thermo Fisher TRIzolReagent User Guide)
5. Incubate for 2-3 minutes. 
SECTION: RNA Isolation (from Thermo Fisher TRIzolReagent User Guide)
6. Centrifuge the sample for 15 minutes at 12,000 x g at 4ºC
The mixture separates into a lower phenol-chloroform, an interphase, and a colorless upper aqueous phase.
SECTION: RNA Isolation (from Thermo Fisher TRIzolReagent User Guide)
7. Transfer the aqueous phase containing the RNA to a new tube by angling the tube at 45° and pipetting the solution out.
SECTION: RNA Purification and Concentration
8.   
*Extracted from Qiagen RNeasy Micro - pgs 54-57
SECTION: Primary Analysis and De-multiplexing
11. Base call (.bcl) files for each cycle of sequencing are generated by Illumina Real Time Analysis (RTA) software. The base call files and run folders are streamed to servers maintained at the Minnesota Supercomputing Institute. Primary analysis and de-multiplexing are performed using Illumina’s bcl2fastq v2.20. The end result of the bcl2fastq workflow is de-multiplexed FASTQ files.
https://support.illumina.com/downloads/bcl2fastq-conversion-software-v2-20.html

SECTION: Library Preparation
9. Sample Quality Assessment: Total eukaryotic RNA isolates are quantified using a fluorimetric RiboGreen assay.  Total RNA integrity is assessed using capillary electrophoresis (e.g. Agilent BioAnalyzer 2100), generating an RNA Integrity Number (RIN).  For samples to pass the initial QC step, they need to quantify higher than 500 ng and have a RIN of 8 or greater.  Total RNA samples are then converted to Illumina sequencing libraries.

Library Creation: Total RNA samples are converted to Illumina sequencing libraries using Illumina’s TruSeq RNA Sample Preparation Kit (Cat. # RS-122-2001 or RS-122-2002) or Stranded mRNA Sample Preparation Kit (Cat. # RS-122-2101).  (Please see www.illumina.com for a detailed list of kit contents and methods). In summary, the mRNA from a normalized input mass of total RNA is isolated using oligo-dT coated magnetic beads, fragmented and then reverse transcribed into cDNA. The cDNA is blunt-ended, A-tailed and indexed by ligating molecularly barcoded adaptors.  Libraries are amplified using 15 cycles of PCR. Final library size distribution is validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen) and via Q-PCR.  Indexed libraries are then normalized, pooled and size-selected to 320 bp (tight) using the PippinHT instrument.