GSM4742413: DNAse_seq-B6xSPRET-B_cell-DS39205A; Mus musculus; DNase-Hypersensitivity
Whole liver (all lobes), both kidneys and all lobes of the lungs were rapidly dissected from sacrificed mice. Tissues were minced in 2 mm square pieces and resuspended in 5 mL of homogenization buffer per tissue. Nuclei were released using 5-10 strokes in a Dounce homogenizer with a loose-fitting type-A pestle and the resulting homogenate was filtered through a 120µm filter. Marrow was obtained from femurs of 8 week old female mice. B cells were isolated using an AutoMACS (Miltenyi Biotech) to deplete CD43 and Mac-1/CD11b markers. Nuclei were extracted by resuspending cells in buffer A supplemented with 0.015% detergent (IGEPAL-CA630) (Sigma) and incubating for 5-10 minutes on ice. Fresh nuclei were digested with DNase I for 3 minutes. Fragments <250 bp were recovered by sucrose ultracentrifugation, end-repaired and ligated with adapters compatible with the Illumina sequencing platform. Libraries were amplified using minimal PCR cycles based on a trial qPCR amplification. Total RNA was isolated using the mirVana miRNA Isolation Kit with phenol (AM1560). Spike-in controls were mixed in (Ambion-ERCC Mix, Cat no. 4456740) and Illumina sequencing libraries were made using the RNA TruSeq Stranded total RNA (Illumina).