GSM6123573: iFoxk1_ND_D3_rep1; Mus musculus; ATAC-seq
iHA-Foxk1 and Foxk1 KO EBs were collected at different time periods during differentiation and disaggregated in 0.25% trypsin at 37C for 3 min incubation with gentle agitation followed by inactivation with culture medium containing 10% FBS. Cells were collected by centrifugation at 500g for 5 minutes at 4 degrees celcius and washed once with ice-cold PBS. ATAC reaction was performed with 50,000 cells as previously described38 using the Tn5 transposase (Illumina) and libraries were created at the University of Minnesota Genome Center. Libraries were then sequenced on a NextSeq Illumina platform (2x50 bp) aiming for 25 million reads per sample. Library Creation:   ATAC-Seq library prep was performed in accordance with the ATAC-Seq protocol as described by Buenrostro, et al. In brief, transposed DNA was amplified for 5 cycles using Nextera-compatible indexed sequencing adapters. 5 ul from the PCR reaction was ran on qPCR to assess the number of additional PCR cycles required without reducing library diversity. All samples underwent additional PCR cycling as determined by their qPCR amplification curve. Final library size distribution is validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen). Libraries are normalized, pooled, and subjected to a double-sided AmPure to deplete un-tagmented material. Pools are QC'ed via Picogreen, Agilent BioAnalyzer High-Sensitivity, and KAPA qPCR (to assess library functionality) before normalization for sequencing. 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, 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.