GSM5620326: KO-TAC-2; Mus musculus; RNA-Seq
The RNA was quantified using a fluorometric RiboGreen assay and RNA integrity was assessed using capillary electrophoresis and generating an RNA Integrity Number (RIN) (Agilent BioAnalyzer 2100). The samples with higher than 500 ng and RIN of 8 or greater were used to generate 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). The libraries were amplified using 15 cycles of PCR and 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. Pooled libraries are denatured and diluted to the appropriate clustering concentration. The libraries are then loaded onto the NovaSeq paired-end flow cell and clustering occurs onboard the instrument. Once clustering is complete, the sequencing reaction immediately begins using Illumina's 2-color SBS chemistry. Upon completion of reading 1, a 7 base pair index read is performed in the case of single indexed libraries. If dual indexing was used during library preparation, 2 separate 8 or 10 base pair index reads are performed. Finally, the clustered library fragments are re-synthesized in the reverse direction thus producing the template for paired-end read 2. Base call 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 that are released to client accounts for subsequent analysis by the mapping software and aligner of their own choosing.