GSM6124497: D3_Foxk1KO_EB_rep2; Mus musculus; RNA-Seq
Total RNA was isolated from D3, D5 and D10 Control (iHA-Foxk1) and Foxk1 KO EBs using the RNeasy kit (Qiagen, Cat# 74104) following the manufacturer's protocol. Briefly, EBs were lysed in RLT-lysis buffer, followed by a column-based purification process and on-column DNA digestion. Libraries for bulk RNAseq were generated by the University of Minnesota Genome Center. 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. Cluster generation and sequencing:  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 on board the instrument.  Once clustering is complete, the sequencing reaction immediately begins using Illumina's 2-color SBS chemistry.  Upon completion of read 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.    Primary analysis and de-multiplexing:  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 that are released to client accounts for subsequent analysis by the mapping software and aligner of their own choosing.